1 00:00:00,500 --> 00:00:02,830 The following content is provided under a Creative 2 00:00:02,830 --> 00:00:04,370 Commons license. 3 00:00:04,370 --> 00:00:06,670 Your support will help MIT OpenCourseWare 4 00:00:06,670 --> 00:00:11,030 continue to offer high-quality educational resources for free. 5 00:00:11,030 --> 00:00:13,660 To make a donation or view additional materials 6 00:00:13,660 --> 00:00:17,610 from hundreds of MIT courses, visit MIT OpenCourseWare 7 00:00:17,610 --> 00:00:18,540 at ocw.mit.edu. 8 00:00:26,045 --> 00:00:27,920 JOANNE STUBBE: OK, so what I want to do today 9 00:00:27,920 --> 00:00:31,670 is hopefully finish up or get pretty close to finishing up 10 00:00:31,670 --> 00:00:37,430 module 6, where we've been focused on bacterial uptake 11 00:00:37,430 --> 00:00:38,855 of iron into cells. 12 00:00:41,888 --> 00:00:47,720 In the last lecture, I briefly introduced you 13 00:00:47,720 --> 00:00:51,700 to gram-positive and gram-negative 14 00:00:51,700 --> 00:00:54,940 big peptidoglycan, small peptidoglycan, 15 00:00:54,940 --> 00:00:56,690 outer-cell membrane. 16 00:00:56,690 --> 00:00:58,017 They both have the same goals. 17 00:00:58,017 --> 00:00:58,850 They've got to get-- 18 00:00:58,850 --> 00:01:02,900 They take up iron the same way from a siderophore, which 19 00:01:02,900 --> 00:01:08,365 is what we talked about last time, or by a heme. 20 00:01:08,365 --> 00:01:09,990 And we'll talk a little bit about that. 21 00:01:09,990 --> 00:01:12,860 And that's what you focused on in your problem set. 22 00:01:12,860 --> 00:01:15,170 But they have different apparati to do 23 00:01:15,170 --> 00:01:19,190 that, because of the differences between the outer-- 24 00:01:19,190 --> 00:01:21,530 because of the cell walls' distinctions 25 00:01:21,530 --> 00:01:24,530 between gram-negative and gram-positive. 26 00:01:24,530 --> 00:01:27,470 So we were talking, at the end of the class, 27 00:01:27,470 --> 00:01:29,930 about, this was for the siderophores 28 00:01:29,930 --> 00:01:30,980 which we talked about. 29 00:01:30,980 --> 00:01:33,290 We need to take them up. 30 00:01:33,290 --> 00:01:36,470 These are common to all uptake systems. 31 00:01:36,470 --> 00:01:41,255 You have some kind of ATPase system and ABC ATPase. 32 00:01:41,255 --> 00:01:43,130 We're not going to talk about that in detail, 33 00:01:43,130 --> 00:01:47,540 but it uses ATP to bring these molecules 34 00:01:47,540 --> 00:01:52,620 and also heme molecules across the plasma membrane. 35 00:01:52,620 --> 00:01:55,340 And then, in all cases, you have this issue 36 00:01:55,340 --> 00:01:58,940 of how do you get the iron out of whatever the carrier is, 37 00:01:58,940 --> 00:02:03,320 be it a siderophore where the carriers can bind very tightly 38 00:02:03,320 --> 00:02:07,130 or heme where you also have to do something 39 00:02:07,130 --> 00:02:12,060 to get the iron out of the heme so that it can be used. 40 00:02:12,060 --> 00:02:17,280 And so what I want to just say, very briefly-- 41 00:02:17,280 --> 00:02:18,680 and this you all should know now. 42 00:02:18,680 --> 00:02:20,570 So now we're looking at heme uptake. 43 00:02:20,570 --> 00:02:24,110 I'm not going to spend a lot of time drawing the pictures out, 44 00:02:24,110 --> 00:02:30,530 but, if you look at the PowerPoint cartoon, what 45 00:02:30,530 --> 00:02:33,890 you will see is there is a protein like this, which 46 00:02:33,890 --> 00:02:36,840 hopefully you now have been introduced to from your problem 47 00:02:36,840 --> 00:02:37,340 set. 48 00:02:37,340 --> 00:02:43,130 So this could be IsdB or IsdH. 49 00:02:43,130 --> 00:02:46,640 And we'll come back to that, subsequently. 50 00:02:46,640 --> 00:02:51,080 And it sits on the outside of the peptidoglycan. 51 00:02:51,080 --> 00:02:53,150 So this is the protein. 52 00:02:53,150 --> 00:02:59,360 The key thing that is present in all these Isd proteins 53 00:02:59,360 --> 00:03:02,840 is-- let me draw this differently-- is a NEAT domain. 54 00:03:05,520 --> 00:03:06,020 OK? 55 00:03:06,020 --> 00:03:07,320 And we'll come back to that later on. 56 00:03:07,320 --> 00:03:08,060 But this domain-- 57 00:03:08,060 --> 00:03:09,643 So you have a big protein, and there's 58 00:03:09,643 --> 00:03:12,860 one little domain that's going to suck the heme out. 59 00:03:12,860 --> 00:03:16,910 And so what happens is we'll see in Staph. aureus, which 60 00:03:16,910 --> 00:03:20,650 is what we're going to be focused on, 61 00:03:20,650 --> 00:03:24,170 you have hemoglobin. 62 00:03:24,170 --> 00:03:28,160 And somehow-- and I'm going to indicate heme 63 00:03:28,160 --> 00:03:31,640 as a ball of orange, with a little planar 64 00:03:31,640 --> 00:03:33,470 thing as the protoporphyrin IX. 65 00:03:33,470 --> 00:03:35,510 OK, are you all with me? 66 00:03:35,510 --> 00:03:38,900 And then somehow this gets sucked out 67 00:03:38,900 --> 00:03:41,620 into the NEAT domain, where-- 68 00:03:41,620 --> 00:03:44,600 And again, all of these gram-positive and gram-negative 69 00:03:44,600 --> 00:03:48,890 systems are slightly different, but in the Staph. aureus system 70 00:03:48,890 --> 00:03:50,390 we'll be talking about today and you 71 00:03:50,390 --> 00:03:52,940 had to think about in the problem set you basically 72 00:03:52,940 --> 00:03:56,960 have a cascade of proteins which have additional NEAT 73 00:03:56,960 --> 00:04:03,470 domains from which, because this is such a large peptidoglycan, 74 00:04:03,470 --> 00:04:08,270 you need to transfer the heme to the plasma-membrane 75 00:04:08,270 --> 00:04:09,120 transporter. 76 00:04:09,120 --> 00:04:13,160 And what's interesting about these systems 77 00:04:13,160 --> 00:04:16,459 and is distinct is that they end up, 78 00:04:16,459 --> 00:04:23,950 they're covalently bound to the peptidoglycan. 79 00:04:23,950 --> 00:04:26,360 And I'm going to indicate peptidoglycan as "PG." 80 00:04:26,360 --> 00:04:29,050 And we'll talk about that reaction today-- 81 00:04:29,050 --> 00:04:32,200 the enzyme that catalyzes those reactions. 82 00:04:32,200 --> 00:04:35,740 And all of these guys end up covalently bound 83 00:04:35,740 --> 00:04:38,410 to the peptidoglycan-- which is distinct from all 84 00:04:38,410 --> 00:04:41,530 of the experiments you looked at in your problem set. 85 00:04:41,530 --> 00:04:44,590 Nobody can figure out how to make the peptidoglycan 86 00:04:44,590 --> 00:04:46,240 with these things covalently bound. 87 00:04:46,240 --> 00:04:51,070 So what you're looking at is a model for the actual process. 88 00:04:51,070 --> 00:04:55,630 OK, so, also-- so that's the gram-positive. 89 00:04:55,630 --> 00:05:01,100 And in the gram-negative, one has two ways of doing this. 90 00:05:01,100 --> 00:05:05,350 And again, these parallel the ways with siderophore uptake. 91 00:05:05,350 --> 00:05:09,770 So you have an outer membrane-- 92 00:05:09,770 --> 00:05:11,890 So this is the outer membrane. 93 00:05:11,890 --> 00:05:18,190 And you have a beta barrel, with a little plug in it. 94 00:05:18,190 --> 00:05:21,070 And so these beta barrels, they're at, like, 20 or 30 95 00:05:21,070 --> 00:05:23,410 of these things in the outer membranes. 96 00:05:23,410 --> 00:05:25,180 And they can take up siderophores, 97 00:05:25,180 --> 00:05:27,700 as we talked about last time, but they can also 98 00:05:27,700 --> 00:05:28,600 take up hemes. 99 00:05:28,600 --> 00:05:29,100 OK? 100 00:05:29,100 --> 00:05:30,750 So each one of these is distinct, 101 00:05:30,750 --> 00:05:34,930 although the structures are all pretty much the same. 102 00:05:34,930 --> 00:05:38,710 And so what you see in this case is, 103 00:05:38,710 --> 00:05:46,360 there are actually two ways that you can take heme up. 104 00:05:46,360 --> 00:05:50,050 So you can take up heme directly. 105 00:05:50,050 --> 00:05:53,320 And we'll see that what we'll be looking at 106 00:05:53,320 --> 00:05:58,180 is hemoglobin, which has four alpha 2 beta 2. 107 00:05:58,180 --> 00:06:00,550 So this could be hemoglobin. 108 00:06:00,550 --> 00:06:03,310 That's one of the major sources, and it is the major source 109 00:06:03,310 --> 00:06:05,470 for Staph. aureus. 110 00:06:05,470 --> 00:06:10,270 And so this can bind directly to the beta barrel-- 111 00:06:10,270 --> 00:06:12,060 gets extracted. 112 00:06:12,060 --> 00:06:13,420 The heme gets extracted. 113 00:06:13,420 --> 00:06:15,250 The protein doesn't get through. 114 00:06:15,250 --> 00:06:21,030 And so the heme is transferred through this beta barrel. 115 00:06:21,030 --> 00:06:22,210 OK. 116 00:06:22,210 --> 00:06:24,130 So that's one mechanism. 117 00:06:24,130 --> 00:06:27,070 And then there's a second mechanism. 118 00:06:27,070 --> 00:06:30,970 And the second mechanism involves a hemophore. 119 00:06:34,800 --> 00:06:40,660 And the hemophore is going to pick up the heme. 120 00:06:40,660 --> 00:06:43,960 And so every organism is distinct. 121 00:06:43,960 --> 00:06:46,690 There are many kinds of hemophores. 122 00:06:46,690 --> 00:06:50,260 And I have a definition of all of these-- 123 00:06:50,260 --> 00:06:52,240 the nomenclature involved. 124 00:06:52,240 --> 00:06:55,600 And so, after class today, I'll update these notes, 125 00:06:55,600 --> 00:06:57,340 because that's not in the original-- 126 00:06:57,340 --> 00:07:01,060 the definitions aren't in the original PowerPoint. 127 00:07:01,060 --> 00:07:01,780 OK? 128 00:07:01,780 --> 00:07:06,220 So what you have, over here, is the hemophore 129 00:07:06,220 --> 00:07:13,240 that somehow extracts the heme out 130 00:07:13,240 --> 00:07:15,370 of hemoglobin or haptoglobin. 131 00:07:15,370 --> 00:07:16,840 We'll see that's another thing. 132 00:07:16,840 --> 00:07:20,650 So this gets extracted and then gets 133 00:07:20,650 --> 00:07:23,290 transferred, in that fashion. 134 00:07:23,290 --> 00:07:27,280 And so these hemophores come in all flavors and shapes. 135 00:07:27,280 --> 00:07:30,590 They're different-- for example, in Pseudomonas or M. 136 00:07:30,590 --> 00:07:31,570 tuberculosis. 137 00:07:31,570 --> 00:07:34,000 And we're not going to talk about them further, 138 00:07:34,000 --> 00:07:39,220 but the idea is they all use these beta-barrel proteins 139 00:07:39,220 --> 00:07:43,630 to be able to somehow transfer the heme across. 140 00:07:43,630 --> 00:07:46,240 And what happens, just as in the case-- if you go back 141 00:07:46,240 --> 00:07:48,340 and you look at your notes from last time, 142 00:07:48,340 --> 00:07:50,740 there's a periplasmic binding protein 143 00:07:50,740 --> 00:07:54,790 that takes the heme and shuttles it, again, 144 00:07:54,790 --> 00:07:57,230 to these ABC transporters. 145 00:07:57,230 --> 00:07:57,730 OK? 146 00:07:57,730 --> 00:08:00,610 So, in this system, again, you have 147 00:08:00,610 --> 00:08:06,260 a periplasmic binding protein. 148 00:08:09,750 --> 00:08:19,000 And this goes to the ABC transporter, 149 00:08:19,000 --> 00:08:25,570 which uses ATP and the energy of hydrolysis of ATP, 150 00:08:25,570 --> 00:08:27,290 to transfer this into the cytosol. 151 00:08:30,090 --> 00:08:31,750 OK, so this is the same. 152 00:08:31,750 --> 00:08:34,210 That remains the same. 153 00:08:34,210 --> 00:08:37,539 And the transporters are distinct. 154 00:08:37,539 --> 00:08:40,240 And then, again, once you get inside the cell, 155 00:08:40,240 --> 00:08:41,590 what do you have to do? 156 00:08:41,590 --> 00:08:43,539 You've got to get the iron out of the heme. 157 00:08:43,539 --> 00:08:46,100 So the problems that you're facing 158 00:08:46,100 --> 00:08:49,540 are very similar to the siderophores. 159 00:08:49,540 --> 00:08:53,030 So, in all cases-- 160 00:08:53,030 --> 00:08:58,340 So the last step is, in the cytosol, 161 00:08:58,340 --> 00:09:05,590 you need to extract the iron. 162 00:09:05,590 --> 00:09:07,030 And you can extract-- 163 00:09:07,030 --> 00:09:10,630 usually, this is in a plus-3 oxidation state. 164 00:09:10,630 --> 00:09:12,910 So you extract the iron. 165 00:09:12,910 --> 00:09:21,350 And this can be done by a heme oxygenase, which 166 00:09:21,350 --> 00:09:23,320 degrades the heme. 167 00:09:23,320 --> 00:09:23,820 OK. 168 00:09:26,960 --> 00:09:29,240 In some cases, people have reported 169 00:09:29,240 --> 00:09:32,510 that you can reduce the iron 3 to iron 2, when the heme can 170 00:09:32,510 --> 00:09:35,570 come out, but that still probably is not an easy task 171 00:09:35,570 --> 00:09:37,400 because you've got four-- 172 00:09:37,400 --> 00:09:40,820 you've got four nitrogens, chelating to the heme, 173 00:09:40,820 --> 00:09:42,890 and the exchange, the ligand exchange, rates 174 00:09:42,890 --> 00:09:44,510 are probably really slow. 175 00:09:44,510 --> 00:09:47,690 So I would say the major way of getting 176 00:09:47,690 --> 00:09:50,630 the iron out of the heme is by degradation of the heme. 177 00:09:50,630 --> 00:09:54,710 And we're not going to talk about that in detail at all, 178 00:09:54,710 --> 00:09:55,250 either. 179 00:09:55,250 --> 00:09:56,450 OK. 180 00:09:56,450 --> 00:09:58,820 So that's the introductory part. 181 00:09:58,820 --> 00:10:00,380 And here's the nomenclature, which 182 00:10:00,380 --> 00:10:01,860 I've already gone through. 183 00:10:01,860 --> 00:10:03,920 I've got all these terms defined. 184 00:10:03,920 --> 00:10:06,710 And if you don't remember that, or you don't remember it 185 00:10:06,710 --> 00:10:09,920 from the reading, you have a page with all the names-- 186 00:10:09,920 --> 00:10:12,050 which are confusing. 187 00:10:12,050 --> 00:10:14,390 And so the final thing I wanted to say, 188 00:10:14,390 --> 00:10:19,550 before we go on and actually start looking at peptidoglycans 189 00:10:19,550 --> 00:10:21,740 and gram-positive bacteria and heme uptake 190 00:10:21,740 --> 00:10:25,310 in Staph. aureus, which is what I was going to focus on 191 00:10:25,310 --> 00:10:29,090 in this little module, is to just show you, 192 00:10:29,090 --> 00:10:32,010 bacteria desperately need iron. 193 00:10:32,010 --> 00:10:33,050 So what do they do? 194 00:10:33,050 --> 00:10:34,700 This is what they do. 195 00:10:34,700 --> 00:10:39,320 OK, so, here you can see-- and some bacteria 196 00:10:39,320 --> 00:10:41,900 make three or four kinds of siderophores. 197 00:10:41,900 --> 00:10:44,870 Others only make one or two kinds of siderophores, 198 00:10:44,870 --> 00:10:46,790 but what they've done is they've figured out 199 00:10:46,790 --> 00:10:51,560 how to scavenge the genes that are required 200 00:10:51,560 --> 00:10:52,970 for these beta barrels. 201 00:10:52,970 --> 00:10:54,830 So they can take up a siderophore 202 00:10:54,830 --> 00:10:57,120 that some other bacteria makes. 203 00:10:57,120 --> 00:10:57,620 OK? 204 00:10:57,620 --> 00:10:59,330 And that's also true of yeast. 205 00:10:59,330 --> 00:11:03,410 Yeast don't make siderophores, but most yeast have, 206 00:11:03,410 --> 00:11:05,870 in their outer membranes, ways of picking up 207 00:11:05,870 --> 00:11:08,708 siderophores and bringing it into the cell, since-- 208 00:11:08,708 --> 00:11:10,250 and remember we talked about the fact 209 00:11:10,250 --> 00:11:13,610 there were 500 different kinds of siderophores. 210 00:11:13,610 --> 00:11:16,890 But you can see that the strategy is exactly the same. 211 00:11:16,890 --> 00:11:18,920 You have a beta barrel. 212 00:11:18,920 --> 00:11:22,670 You have-- these are all periplasmic binding proteins. 213 00:11:22,670 --> 00:11:26,840 This picture is screwed up, in that they forgot the TonB. 214 00:11:26,840 --> 00:11:31,250 Remember, there's a three-component machine, 215 00:11:31,250 --> 00:11:37,100 TonB, ExbB and D, which is connected to a proton motive 216 00:11:37,100 --> 00:11:39,680 force across a plasma membrane, which 217 00:11:39,680 --> 00:11:44,630 is key for getting either the heme or the iron 218 00:11:44,630 --> 00:11:46,860 into the periplasm. 219 00:11:46,860 --> 00:11:49,830 And you use a periplasmic binding protein, 220 00:11:49,830 --> 00:11:54,410 which then goes through these ATPase transp-- 221 00:11:54,410 --> 00:11:57,230 ABC-ATPase transporters. 222 00:11:57,230 --> 00:12:02,090 So what I showed you was heme uptake, iron uptake, but in all 223 00:12:02,090 --> 00:12:06,380 of these cases, like Staph. aureus we'll be talking about, 224 00:12:06,380 --> 00:12:08,590 we can also get iron out of transferrin. 225 00:12:08,590 --> 00:12:09,590 We've talked about that. 226 00:12:09,590 --> 00:12:11,660 That's the major carrier in humans. 227 00:12:11,660 --> 00:12:15,830 The siderophores can actually extract the iron 228 00:12:15,830 --> 00:12:16,940 from the transferrin. 229 00:12:16,940 --> 00:12:19,130 And remember the KD was 10 to the minus 3, 230 00:12:19,130 --> 00:12:22,400 so somehow, again, you've got to get iron transferred 231 00:12:22,400 --> 00:12:24,410 under those conditions. 232 00:12:24,410 --> 00:12:26,090 And that's how these guys survive. 233 00:12:26,090 --> 00:12:32,140 So they're pretty desperate to get iron. 234 00:12:32,140 --> 00:12:34,340 And inside, once they get inside the cell, 235 00:12:34,340 --> 00:12:38,137 you have all variations of the theme to get the iron out. 236 00:12:38,137 --> 00:12:39,470 But they're all sort of similar. 237 00:12:39,470 --> 00:12:42,012 Somehow, you've got to get rid of whatever is tightly binding 238 00:12:42,012 --> 00:12:43,070 it. 239 00:12:43,070 --> 00:12:46,370 And if you're creative, you can reuse 240 00:12:46,370 --> 00:12:50,010 whatever is tightly binding it, to go pick up some more metal. 241 00:12:50,010 --> 00:12:50,510 OK. 242 00:12:50,510 --> 00:12:53,630 So that just summarizes what I just said. 243 00:12:53,630 --> 00:12:56,060 And so, in two seconds, I'm going to show you, 244 00:12:56,060 --> 00:12:57,740 now-- we've spent one whole lecture, 245 00:12:57,740 --> 00:12:59,282 a little more than a lecture, talking 246 00:12:59,282 --> 00:13:03,650 about iron uptake in humans via DMT1, 247 00:13:03,650 --> 00:13:08,900 the iron-2 transporter, and the transferrin transfer receptor. 248 00:13:08,900 --> 00:13:11,390 So, in the plus-two and plus-three states, 249 00:13:11,390 --> 00:13:15,500 we just started looking at the strategies by bacteria 250 00:13:15,500 --> 00:13:18,560 and saw how widespread they are. 251 00:13:18,560 --> 00:13:22,700 And then the question is, how do you win? 252 00:13:22,700 --> 00:13:26,000 OK, bacteria need iron. 253 00:13:26,000 --> 00:13:27,530 We need iron. 254 00:13:27,530 --> 00:13:29,750 And the question is, how do you reach-- 255 00:13:29,750 --> 00:13:31,950 and we have a lot of bacteria growing in us, [LAUGH] 256 00:13:31,950 --> 00:13:35,210 so we've reached some kind of homeostasis. 257 00:13:35,210 --> 00:13:37,010 But with the pathogenic ones, of course, 258 00:13:37,010 --> 00:13:39,050 we really want to get rid of them. 259 00:13:39,050 --> 00:13:40,640 And so that's what the issue is. 260 00:13:40,640 --> 00:13:44,030 And there have been a bunch of articles. 261 00:13:44,030 --> 00:13:46,010 You can read about this in a lot of detail, 262 00:13:46,010 --> 00:13:50,270 if you're interested in the more medical aspects of this. 263 00:13:50,270 --> 00:13:53,570 But this war between bacteria and humans. 264 00:13:53,570 --> 00:13:56,230 And really it's sort of fight for nutrients. 265 00:13:56,230 --> 00:13:59,450 And, in this case, the nutrient is iron. 266 00:13:59,450 --> 00:14:03,050 Has received a lot of attention, because we're 267 00:14:03,050 --> 00:14:05,870 desperate for new kinds of targets 268 00:14:05,870 --> 00:14:08,720 for antibiotics, because of the resistance problems. 269 00:14:08,720 --> 00:14:14,150 And so nutrient limitation and iron sequestration 270 00:14:14,150 --> 00:14:18,288 from a pathogenic organism might represent a new target. 271 00:14:18,288 --> 00:14:19,580 Of course, what are the issues? 272 00:14:19,580 --> 00:14:22,040 The issues are, we also need iron. 273 00:14:22,040 --> 00:14:24,457 And so, if you lower the amount of iron, 274 00:14:24,457 --> 00:14:26,040 then you might be in trouble, as well. 275 00:14:26,040 --> 00:14:29,860 So what we know is, bacteria, viruses-- 276 00:14:29,860 --> 00:14:32,130 bacteria have been extensively studied; 277 00:14:32,130 --> 00:14:40,080 viruses, less so, also protozoa, such as the malaria system-- 278 00:14:40,080 --> 00:14:44,160 all are known to depend on iron for growth. 279 00:14:44,160 --> 00:14:47,370 And so, again, if you want to read about this, 280 00:14:47,370 --> 00:14:49,620 you can read about some of the strategies 281 00:14:49,620 --> 00:14:52,290 these organisms [LAUGH] use to get iron away 282 00:14:52,290 --> 00:14:54,443 from the human systems. 283 00:14:54,443 --> 00:14:55,860 And it's sort of amazing, when you 284 00:14:55,860 --> 00:14:58,320 look at the details of how things have evolved, 285 00:14:58,320 --> 00:15:03,010 back and forth, back and forth, [LAUGH] in terms of survival. 286 00:15:03,010 --> 00:15:05,980 And so really what it's all about is homeostasis. 287 00:15:05,980 --> 00:15:06,810 OK? 288 00:15:06,810 --> 00:15:09,100 And that's what was all about in cholesterol. 289 00:15:09,100 --> 00:15:11,430 And we'll see, with reactive oxygen species, 290 00:15:11,430 --> 00:15:13,650 that's what it's all about. 291 00:15:13,650 --> 00:15:20,020 So, somehow, using hepcidin-- 292 00:15:20,020 --> 00:15:21,900 which is the human master regulator, 293 00:15:21,900 --> 00:15:23,670 the peptide hormone-- 294 00:15:23,670 --> 00:15:26,190 we need to figure out how to keep ourselves alive 295 00:15:26,190 --> 00:15:30,180 while killing off these bacteria, in some way, 296 00:15:30,180 --> 00:15:34,070 by sequestering the iron from the bacteria. 297 00:15:34,070 --> 00:15:34,570 OK. 298 00:15:34,570 --> 00:15:37,050 So this is an important problem that has 299 00:15:37,050 --> 00:15:38,370 received a lot of attention. 300 00:15:38,370 --> 00:15:42,570 And most of you know that the Nolan Lab is doing 301 00:15:42,570 --> 00:15:44,940 beautiful studies in this area. 302 00:15:44,940 --> 00:15:45,690 OK. 303 00:15:45,690 --> 00:15:48,390 So what I want to do now, for the rest of the lecture, 304 00:15:48,390 --> 00:15:50,430 is focus on Staph. aureus. 305 00:15:50,430 --> 00:15:51,960 OK? 306 00:15:51,960 --> 00:15:57,270 And Staph. aureus is-- 307 00:15:57,270 --> 00:16:02,550 methicillin-resistant Staph. aureus is a major problem, 308 00:16:02,550 --> 00:16:04,050 throughout the world. 309 00:16:04,050 --> 00:16:06,240 We don't have any ways to kill this guy. 310 00:16:06,240 --> 00:16:08,880 And so that's why I decided to pick this target, 311 00:16:08,880 --> 00:16:12,240 but there are many other [LAUGH] of these pathogens around that 312 00:16:12,240 --> 00:16:14,460 have problems-- 313 00:16:14,460 --> 00:16:18,630 have also resistance problems, Staph. aureus 314 00:16:18,630 --> 00:16:21,120 being the one that's been most extensively studied 315 00:16:21,120 --> 00:16:24,000 in the last decade or so. 316 00:16:24,000 --> 00:16:28,100 But bacteria has come back in vogue. 317 00:16:28,100 --> 00:16:31,570 For years, nobody on campus cared anything about [LAUGH] 318 00:16:31,570 --> 00:16:33,540 microorganisms or bacteria. 319 00:16:33,540 --> 00:16:36,283 The microbiome has brought it back in vogue, 320 00:16:36,283 --> 00:16:37,950 because people think they're going to be 321 00:16:37,950 --> 00:16:39,240 able to figure that all out. 322 00:16:39,240 --> 00:16:40,260 OK. 323 00:16:40,260 --> 00:16:43,880 But anyhow, bacteria have always been extremely important, 324 00:16:43,880 --> 00:16:45,540 not only in terms of human health 325 00:16:45,540 --> 00:16:49,002 but in terms of how the whole world functions. 326 00:16:49,002 --> 00:16:50,460 There are so many of them, and they 327 00:16:50,460 --> 00:16:52,680 do so much interesting stuff. 328 00:16:52,680 --> 00:16:54,690 And we have to live with them, side by side. 329 00:16:54,690 --> 00:16:58,568 So anyhow, we're going to look at Staph. aureus. 330 00:16:58,568 --> 00:17:01,110 That's what we're going to focus on, because of this problem. 331 00:17:01,110 --> 00:17:04,650 And I think Staph. aureus, which many people don't realize, 332 00:17:04,650 --> 00:17:08,940 is that 30% of all people have Staph. aureus on your skin 333 00:17:08,940 --> 00:17:13,690 or in regions that are not breaching into the bloodstream. 334 00:17:13,690 --> 00:17:15,560 So we all have Staph. aureus. 335 00:17:15,560 --> 00:17:21,300 So 30% of us have this bacteria. 336 00:17:21,300 --> 00:17:25,560 If you get-- if wherever it's localized is breached, 337 00:17:25,560 --> 00:17:28,349 and it gets into our bloodstream, 338 00:17:28,349 --> 00:17:30,240 then it's all over, because Staph. aureus 339 00:17:30,240 --> 00:17:31,890 can colonize almost anywhere. 340 00:17:31,890 --> 00:17:33,870 That's different from other organisms. 341 00:17:33,870 --> 00:17:36,300 Some organisms can only colonize in the lungs. 342 00:17:36,300 --> 00:17:38,200 Some colonize in the heart. 343 00:17:38,200 --> 00:17:44,590 So these can colonize almost all tissues. 344 00:17:44,590 --> 00:17:46,320 And what you know is, if you start 345 00:17:46,320 --> 00:17:49,320 thinking about physiology-- and again, I'm not an MD-- 346 00:17:49,320 --> 00:17:52,270 but different tissues have different environments. 347 00:17:52,270 --> 00:17:52,900 OK? 348 00:17:52,900 --> 00:17:55,650 And so a lot of organisms find siderophore an environment 349 00:17:55,650 --> 00:18:01,260 where they can best live and then take up-- 350 00:18:01,260 --> 00:18:02,730 make their home there. 351 00:18:02,730 --> 00:18:04,590 But Staph. aureus is one of these guys 352 00:18:04,590 --> 00:18:07,050 that can go anywhere. 353 00:18:07,050 --> 00:18:12,410 And so this makes it specifically very insidious. 354 00:18:12,410 --> 00:18:18,560 And you can get septicemia, or you can get endocarditis, 355 00:18:18,560 --> 00:18:22,520 or you can get all kinds of horrible diseases associated 356 00:18:22,520 --> 00:18:26,310 with Staph. aureus, once it breaches the barrier. 357 00:18:26,310 --> 00:18:26,810 OK. 358 00:18:26,810 --> 00:18:31,850 So what we need to do, as you've already seen from your problem 359 00:18:31,850 --> 00:18:38,020 set, to understand how Staph. aureus can get heme 360 00:18:38,020 --> 00:18:45,570 into its cytosol to be able to function, 361 00:18:45,570 --> 00:18:48,320 to be able to grow effectively, is, 362 00:18:48,320 --> 00:18:51,380 we need to look at the outer cell wall or the peptidoglycan. 363 00:18:51,380 --> 00:18:55,370 So what I'm going to do is spend a few minutes 364 00:18:55,370 --> 00:18:58,520 talking about the structure of the peptidoglycan. 365 00:18:58,520 --> 00:19:00,140 And then we'll go back in and we'll 366 00:19:00,140 --> 00:19:04,250 talk about how these proteins that you worked on 367 00:19:04,250 --> 00:19:07,390 in the problem set covalently bind to the peptidoglycan 368 00:19:07,390 --> 00:19:09,920 and allow you to take up iron to the cell. 369 00:19:09,920 --> 00:19:13,250 And why is heme a major target? 370 00:19:13,250 --> 00:19:15,740 Heme is a major target for Staph. aureus. 371 00:19:15,740 --> 00:19:17,420 They've evolved. 372 00:19:17,420 --> 00:19:19,810 The major source of iron, we all know, 373 00:19:19,810 --> 00:19:22,580 is hemoglobin now, in red blood cells. 374 00:19:22,580 --> 00:19:26,270 And so Staph. aureus has developed proteins-- 375 00:19:26,270 --> 00:19:29,150 endotoxins, really-- that can go in 376 00:19:29,150 --> 00:19:34,070 and-- there's proteins that can insert into red blood cell 377 00:19:34,070 --> 00:19:36,580 membranes, make a pore. 378 00:19:36,580 --> 00:19:40,730 The blood cells lyse, and now the bacteria 379 00:19:40,730 --> 00:19:43,640 are extremely happy because they have huge amounts of heme. 380 00:19:43,640 --> 00:19:47,930 And then they want to take that heme into-- 381 00:19:47,930 --> 00:19:49,320 to help them survive. 382 00:19:49,320 --> 00:19:52,370 So Staph. aureus are amazingly creative, 383 00:19:52,370 --> 00:19:57,610 in terms of getting the heme that they need for survival. 384 00:19:57,610 --> 00:19:58,110 OK. 385 00:19:58,110 --> 00:20:00,380 So, peptidoglycan. 386 00:20:00,380 --> 00:20:03,440 Most of you have probably seen peptidoglycan before. 387 00:20:03,440 --> 00:20:07,590 I'm just going to say a few things about peptidoglycan. 388 00:20:07,590 --> 00:20:09,700 So let's look at-- 389 00:20:09,700 --> 00:20:10,200 let's see. 390 00:20:10,200 --> 00:20:11,190 Where do I want to do this? 391 00:20:11,190 --> 00:20:11,690 All right. 392 00:20:11,690 --> 00:20:13,060 So I'm going to erase this. 393 00:20:13,060 --> 00:20:15,900 We're going to look at the cell wall. 394 00:20:15,900 --> 00:20:16,400 OK. 395 00:20:20,120 --> 00:20:23,150 And what you can see, here, I'm going to draw just a few things 396 00:20:23,150 --> 00:20:24,030 on the board. 397 00:20:24,030 --> 00:20:26,810 But what you can see here, in this cartoon, 398 00:20:26,810 --> 00:20:29,720 is you have two kinds of sugars-- 399 00:20:29,720 --> 00:20:33,500 N-acetylglucosamine and N-acetylmuramic acid. 400 00:20:33,500 --> 00:20:36,020 N-acetylglucosamine is a precursor 401 00:20:36,020 --> 00:20:38,780 to N-acetylmuramic acid. 402 00:20:38,780 --> 00:20:42,530 And what you see, attached to N-acetylmuramic acid, 403 00:20:42,530 --> 00:20:44,270 are little blue balls. 404 00:20:44,270 --> 00:20:46,280 And that's the peptide that turns out 405 00:20:46,280 --> 00:20:47,840 it starts out with a pentapeptide 406 00:20:47,840 --> 00:20:50,540 and goes to a tetrapeptide. 407 00:20:50,540 --> 00:20:53,520 And what you see here, in the purple balls-- 408 00:20:53,520 --> 00:20:57,170 and this is unique to Staph. aureus-- 409 00:20:57,170 --> 00:21:00,330 is, other amino acids, they're all the same, 410 00:21:00,330 --> 00:21:01,250 and this is glycine. 411 00:21:01,250 --> 00:21:06,080 So, if you look down here, here are the disaccharides, shown up 412 00:21:06,080 --> 00:21:07,430 here. 413 00:21:07,430 --> 00:21:10,670 Here is-- yeah, one, two, three, four, five. 414 00:21:10,670 --> 00:21:12,870 Here is the pentapeptide. 415 00:21:12,870 --> 00:21:16,800 And what do you notice unusual about the pentapeptide? 416 00:21:16,800 --> 00:21:19,340 You have a D glutamine. 417 00:21:19,340 --> 00:21:20,060 OK? 418 00:21:20,060 --> 00:21:22,580 And I was just reading a whole bunch of papers 419 00:21:22,580 --> 00:21:23,660 on somebody's thesis-- 420 00:21:23,660 --> 00:21:26,012 tomorrow, actually. 421 00:21:26,012 --> 00:21:27,470 And you're trying to make this guy, 422 00:21:27,470 --> 00:21:28,920 nobody can study this stuff. 423 00:21:28,920 --> 00:21:29,630 Why? 424 00:21:29,630 --> 00:21:32,550 Because you have to make a peptidoglycan. 425 00:21:32,550 --> 00:21:33,300 And I'll show you. 426 00:21:33,300 --> 00:21:34,130 It's complicated. 427 00:21:34,130 --> 00:21:36,360 You have to stick on a pentapeptide. 428 00:21:36,360 --> 00:21:38,905 You have to stick on the glycines. 429 00:21:38,905 --> 00:21:40,280 And how do you get the substrates 430 00:21:40,280 --> 00:21:42,110 for your enzymatic reactions? 431 00:21:42,110 --> 00:21:44,570 So we've known this pathway for decades, 432 00:21:44,570 --> 00:21:46,190 but it's taken really good chemists 433 00:21:46,190 --> 00:21:50,660 to be able to figure out how to look at these individual steps. 434 00:21:50,660 --> 00:21:54,740 And so what's unusual, here, is, if you replace glutamine 435 00:21:54,740 --> 00:21:58,490 with a glutamate, it doesn't work very well at all. 436 00:21:58,490 --> 00:22:00,530 OK, so it's that subtle. 437 00:22:00,530 --> 00:22:02,840 Here you've got this huge macromolecule, 438 00:22:02,840 --> 00:22:05,660 and you're replacing an NH2 with an OH, 439 00:22:05,660 --> 00:22:09,920 and you alter the resistance to different bacteria. 440 00:22:09,920 --> 00:22:13,130 And again, you have this unusual pentaglycine. 441 00:22:13,130 --> 00:22:16,610 And you'll see in the cartoon, in a few minutes, 442 00:22:16,610 --> 00:22:20,280 where do you think this glycine, pentaglycine comes from? 443 00:22:20,280 --> 00:22:23,910 Well, it actually comes from a tRNA that binds glycine. 444 00:22:23,910 --> 00:22:25,980 OK, you've seen that before. 445 00:22:25,980 --> 00:22:28,430 But, instead of using the ribosome 446 00:22:28,430 --> 00:22:33,230 to make this little peptide, it uses nonribosomal peptide 447 00:22:33,230 --> 00:22:34,520 synthetases. 448 00:22:34,520 --> 00:22:37,520 And this all happens in the cytosol of the cell. 449 00:22:37,520 --> 00:22:41,300 So, what do we know about the structure? 450 00:22:41,300 --> 00:22:47,330 I'm just going to draw N-acetylglucosamine. 451 00:22:47,330 --> 00:22:51,680 And what I'm going to do is put some R groups on here. 452 00:22:51,680 --> 00:22:53,960 So I'm going to put OX. 453 00:22:53,960 --> 00:22:57,680 And then here we have N-acetyl. 454 00:22:57,680 --> 00:23:01,040 So that's an acetate group. 455 00:23:01,040 --> 00:23:03,545 Here I'm going to put another OR group. 456 00:23:08,740 --> 00:23:13,600 OK, so the two things I want to focus on, 457 00:23:13,600 --> 00:23:17,800 the two things I'm going to focus on, is this X 458 00:23:17,800 --> 00:23:21,677 and this R. So is N-acetylglucosamine. 459 00:23:24,790 --> 00:23:31,570 And then the second one is N-acetylmuramic acid. 460 00:23:31,570 --> 00:23:39,172 And, in both of these cases, X is equal to UDP. 461 00:23:39,172 --> 00:23:40,630 So we're going to come back to this 462 00:23:40,630 --> 00:23:43,120 in the last module on nucleotides. 463 00:23:43,120 --> 00:23:47,620 So nucleotides play a central role in RNA and DNA, 464 00:23:47,620 --> 00:23:49,750 but they also play a central role 465 00:23:49,750 --> 00:23:53,290 in moving around all sugars inside the cell. 466 00:23:53,290 --> 00:23:57,640 So what you have here, actually, is a pyrophosphate linkage 467 00:23:57,640 --> 00:23:59,130 to UDP. 468 00:23:59,130 --> 00:24:00,430 OK? 469 00:24:00,430 --> 00:24:04,370 And if we look at N-acetylglucosamine, 470 00:24:04,370 --> 00:24:07,360 R is equal to H. OK? 471 00:24:07,360 --> 00:24:10,720 But if we look at muramic acid, what we're going to see 472 00:24:10,720 --> 00:24:15,565 is that nature has put on a lactic acid in this position. 473 00:24:18,780 --> 00:24:23,040 OK, so here's your methyl group, from your lactic acid. 474 00:24:23,040 --> 00:24:24,860 And here's the carboxylate. 475 00:24:24,860 --> 00:24:32,570 So this is the R group in N-acetylmuramic acid. 476 00:24:32,570 --> 00:24:33,440 OK. 477 00:24:33,440 --> 00:24:35,930 Now, what we're going to see is, while most sugars-- 478 00:24:35,930 --> 00:24:41,390 and this is true in humans, and it's also true in bacteria-- 479 00:24:41,390 --> 00:24:44,540 are carried around and transported within the cell 480 00:24:44,540 --> 00:24:48,770 as linked nucleotides, what we'll also see in the cell 481 00:24:48,770 --> 00:24:53,690 wall-- which has made them extremely challenging to study, 482 00:24:53,690 --> 00:24:57,380 made the whole pathway extremely challenging to study-- 483 00:24:57,380 --> 00:25:03,200 in addition to X equal to UDP, X can also 484 00:25:03,200 --> 00:25:11,240 be equal to sort of an amazing structure. 485 00:25:11,240 --> 00:25:13,120 And the structure is slightly different 486 00:25:13,120 --> 00:25:16,490 in different bacteria, but this strategy 487 00:25:16,490 --> 00:25:22,820 is also used in humans, where you have a lipid 488 00:25:22,820 --> 00:25:26,225 and you have a lipid that acts as-- 489 00:25:29,390 --> 00:25:31,820 is made from-- hopefully you now know-- 490 00:25:31,820 --> 00:25:34,120 is isopentenyl pyrophosphate. 491 00:25:34,120 --> 00:25:35,610 OK? 492 00:25:35,610 --> 00:25:37,730 And there are seven of these, where you 493 00:25:37,730 --> 00:25:40,460 have the trans configuration. 494 00:25:40,460 --> 00:25:42,710 There are now three of these, which 495 00:25:42,710 --> 00:25:45,290 have the cis configuration. 496 00:25:48,260 --> 00:25:51,895 Just make sure I get my-- 497 00:25:51,895 --> 00:25:54,190 is that right? 498 00:25:54,190 --> 00:25:55,192 Yeah, that's right. 499 00:25:55,192 --> 00:25:56,650 OK, so you have three of these that 500 00:25:56,650 --> 00:25:58,520 have the cis configuration. 501 00:25:58,520 --> 00:26:02,770 And then you have a terminal dimethyl L configuration. 502 00:26:02,770 --> 00:26:05,590 And this is C55. 503 00:26:05,590 --> 00:26:07,840 So, if you're a synthetic chemist, 504 00:26:07,840 --> 00:26:11,860 and you're trying to stick on a couple of these sugars 505 00:26:11,860 --> 00:26:14,200 with hydrocarbon on the tail, with C55, 506 00:26:14,200 --> 00:26:17,590 you can imagine you would have one heck of a trouble, number 507 00:26:17,590 --> 00:26:20,890 one, synthesizing it but, number two, dealing with it. 508 00:26:20,890 --> 00:26:23,200 And so this goes to the question which 509 00:26:23,200 --> 00:26:24,850 I think is really interesting is, 510 00:26:24,850 --> 00:26:27,880 many people think about polymerization reactions. 511 00:26:27,880 --> 00:26:31,240 We're going to see this polymer is non-template-dependent, 512 00:26:31,240 --> 00:26:35,320 in contrast to polymers of DNA RA, where you have a template. 513 00:26:35,320 --> 00:26:38,990 And furthermore, DNA and RNA are pretty soluble. 514 00:26:38,990 --> 00:26:40,930 These things become insoluble. 515 00:26:40,930 --> 00:26:45,220 So you're making a phase transition from soluble state 516 00:26:45,220 --> 00:26:48,250 to an insoluble state, around the bacteria. 517 00:26:48,250 --> 00:26:52,060 And I think it's really sort of a tribute to Strominger, who 518 00:26:52,060 --> 00:26:54,370 worked on this many years ago, that he figured out 519 00:26:54,370 --> 00:26:55,460 sort of the pathway. 520 00:26:55,460 --> 00:26:59,050 But now it's only with recent studies, and really 521 00:26:59,050 --> 00:27:01,060 some very hard work synthetically, 522 00:27:01,060 --> 00:27:07,240 and also in terms of the microbiology and biochemistry, 523 00:27:07,240 --> 00:27:10,120 that it's really allowed us to elucidate this. 524 00:27:10,120 --> 00:27:16,480 So X, in this case, can also be this lipid. 525 00:27:16,480 --> 00:27:20,000 So I'm just pointing out what the issues are. 526 00:27:20,000 --> 00:27:23,450 And if you look at the cell wall, biosynthetic pathway-- 527 00:27:23,450 --> 00:27:25,840 so this is inside-- 528 00:27:25,840 --> 00:27:28,000 you're not going to be responsible for the details 529 00:27:28,000 --> 00:27:29,260 of this. 530 00:27:29,260 --> 00:27:31,930 But this is outside. 531 00:27:31,930 --> 00:27:32,430 OK. 532 00:27:32,430 --> 00:27:34,850 So you start out with a couple of sugars. 533 00:27:34,850 --> 00:27:37,960 These are the sugars we just talked about. 534 00:27:37,960 --> 00:27:38,480 OK. 535 00:27:38,480 --> 00:27:43,230 So now what you do is add on these five amino acids. 536 00:27:43,230 --> 00:27:45,440 So, over here, we ultimately need 537 00:27:45,440 --> 00:27:51,440 to add on five amino acids. 538 00:27:51,440 --> 00:27:54,380 And what do we see about the amino acids? 539 00:27:54,380 --> 00:27:57,470 They're unusual, because they can have the D-- they are not 540 00:27:57,470 --> 00:27:59,210 necessarily L-amino acids. 541 00:27:59,210 --> 00:28:01,490 They can be D-amino acids. 542 00:28:01,490 --> 00:28:03,170 And these things unfortunately are 543 00:28:03,170 --> 00:28:05,910 unique to different organisms. 544 00:28:05,910 --> 00:28:08,030 So, if you worked out a synthetic method for one, 545 00:28:08,030 --> 00:28:11,180 you're still faced with the problem that every one of them 546 00:28:11,180 --> 00:28:15,480 has different pentapeptides stuck on the end of it. 547 00:28:15,480 --> 00:28:17,180 Now, how would you attach-- 548 00:28:17,180 --> 00:28:20,420 you've now had a lot of biochemistry, 549 00:28:20,420 --> 00:28:21,950 where you've dealt with amino acids, 550 00:28:21,950 --> 00:28:23,720 in the first half of this course. 551 00:28:23,720 --> 00:28:27,770 How would you attach amino acids-- 552 00:28:27,770 --> 00:28:29,570 form and the linkages-- 553 00:28:29,570 --> 00:28:31,430 to this lactic acid? 554 00:28:31,430 --> 00:28:33,670 Can anybody tell me? 555 00:28:33,670 --> 00:28:36,312 What would you do, to make that attachment? 556 00:28:39,756 --> 00:28:43,690 AUDIENCE: You activate the carboxylate. 557 00:28:43,690 --> 00:28:46,190 JOANNE STUBBE: Yeah, so we have to activate the carboxylate. 558 00:28:46,190 --> 00:28:49,364 How do you activate the carboxylate? 559 00:28:49,364 --> 00:28:50,317 AUDIENCE: Make an AMP. 560 00:28:50,317 --> 00:28:51,150 JOANNE STUBBE: Yeah. 561 00:28:51,150 --> 00:28:54,350 So you make an AMP, just like you've seen with nonribosomal-- 562 00:28:54,350 --> 00:28:57,430 the adenlyating enzyme of nonribosomal polypeptide 563 00:28:57,430 --> 00:29:01,510 synthases, and you've seen with tRNAs. 564 00:29:01,510 --> 00:29:02,010 OK. 565 00:29:02,010 --> 00:29:05,920 So you see the same thing, over and over and over again. 566 00:29:05,920 --> 00:29:08,430 So you add these things on. 567 00:29:08,430 --> 00:29:11,310 The difference is that, again, these things, which 568 00:29:11,310 --> 00:29:13,770 are all soluble, down here, these are all 569 00:29:13,770 --> 00:29:15,870 soluble with the nucleotides. 570 00:29:15,870 --> 00:29:18,870 Now, because ultimately this needs 571 00:29:18,870 --> 00:29:20,370 to go from the inside of the cell 572 00:29:20,370 --> 00:29:23,670 to the outside of the cell, what you do, 573 00:29:23,670 --> 00:29:28,230 presumably, is take this lipid-- so you have the C55 lipid, 574 00:29:28,230 --> 00:29:30,690 with one phosphate on it. 575 00:29:30,690 --> 00:29:34,770 And then you attach it to one sugar. 576 00:29:34,770 --> 00:29:36,780 So here it's attached to the muramic acid, 577 00:29:36,780 --> 00:29:39,900 and that's called "lipid 1." 578 00:29:39,900 --> 00:29:43,110 You add N-acetylglucosamine with a glycosyltransferase. 579 00:29:43,110 --> 00:29:44,460 That's lipid 2. 580 00:29:44,460 --> 00:29:47,350 And that's the substrate for the polymerization reaction. 581 00:29:47,350 --> 00:29:48,390 What is the issue? 582 00:29:48,390 --> 00:29:50,760 The issue is, it's in the cytosol 583 00:29:50,760 --> 00:29:54,840 and all the chemistry happens on the outside of the cell. 584 00:29:54,840 --> 00:29:58,500 But, of course, if you move it from the inside to the outside, 585 00:29:58,500 --> 00:30:01,170 you don't want your substrates to float away. 586 00:30:01,170 --> 00:30:02,570 You've got to keep them there. 587 00:30:02,570 --> 00:30:03,120 OK? 588 00:30:03,120 --> 00:30:04,710 And that's especially [LAUGH] true 589 00:30:04,710 --> 00:30:09,040 in gram-positives, where we have no outer membrane. 590 00:30:09,040 --> 00:30:15,890 So the question is, how does this species get from this side 591 00:30:15,890 --> 00:30:18,280 to this side? 592 00:30:18,280 --> 00:30:18,780 OK. 593 00:30:18,780 --> 00:30:21,120 In the last couple years, people have proposed-- 594 00:30:21,120 --> 00:30:22,740 and so this has taken a long time. 595 00:30:22,740 --> 00:30:25,300 People have been looking for these proteins for decades. 596 00:30:25,300 --> 00:30:27,210 These are called "flipases." 597 00:30:27,210 --> 00:30:29,010 So you still have this issue-- again, 598 00:30:29,010 --> 00:30:32,808 this big, huge thing that needs to be transferred. 599 00:30:32,808 --> 00:30:34,350 And I think what's even more amazing, 600 00:30:34,350 --> 00:30:36,840 in the case of Staph. aureus, is that you 601 00:30:36,840 --> 00:30:39,270 put on the pentaglycine in the cytosol. 602 00:30:39,270 --> 00:30:43,050 So, here, what you'll see-- 603 00:30:43,050 --> 00:30:44,050 I think this is E. coli. 604 00:30:44,050 --> 00:30:45,760 I can't remember one from the other. 605 00:30:45,760 --> 00:30:51,190 But, instead of having DAP, which is diaminopimelate, 606 00:30:51,190 --> 00:30:52,680 you actually have lysine. 607 00:30:52,680 --> 00:30:58,020 So, here, what you have in Staph. aureus is a lysine, 608 00:30:58,020 --> 00:31:00,910 and the lysine has an amino group. 609 00:31:00,910 --> 00:31:06,182 And attached to this amino group is the pentaglycine. 610 00:31:06,182 --> 00:31:07,640 And this all occurs in the cytosol. 611 00:31:11,650 --> 00:31:13,790 So this is quite remarkable. 612 00:31:13,790 --> 00:31:20,260 So then, not only do you have to get the disaccharide 613 00:31:20,260 --> 00:31:22,390 with the pentapeptide on it, you need to have, 614 00:31:22,390 --> 00:31:24,650 here, the pentaglycine on it, as well. 615 00:31:24,650 --> 00:31:27,550 And this becomes really important in thinking 616 00:31:27,550 --> 00:31:32,910 about trying to study what's going on in the polymerization 617 00:31:32,910 --> 00:31:37,390 reaction, which is the target of natural products 618 00:31:37,390 --> 00:31:39,160 that are currently used, clinically. 619 00:31:39,160 --> 00:31:39,700 OK. 620 00:31:39,700 --> 00:31:41,680 So this thing's got to flip. 621 00:31:41,680 --> 00:31:43,840 And then what you have is a substrate. 622 00:31:43,840 --> 00:31:45,790 You have a growing chain. 623 00:31:45,790 --> 00:31:49,600 OK, and then what you need to do is extend this chain, 624 00:31:49,600 --> 00:31:51,440 so you have a glycosyltransferase. 625 00:31:51,440 --> 00:31:52,510 So you have two things. 626 00:31:52,510 --> 00:31:56,452 You have phosphoglycosyltransferase. 627 00:32:01,060 --> 00:32:04,540 And then the other thing you have is a TP, which 628 00:32:04,540 --> 00:32:05,860 is a transpeptidase. 629 00:32:10,280 --> 00:32:10,960 OK. 630 00:32:10,960 --> 00:32:13,150 And so the transpeptidase-- we're going to come back 631 00:32:13,150 --> 00:32:14,800 to this in a second, but-- 632 00:32:14,800 --> 00:32:19,390 is ultimately responsible for making a cross link. 633 00:32:19,390 --> 00:32:24,010 Which is what gives the bacteria cell wall rigidity. 634 00:32:24,010 --> 00:32:28,360 Now, in many organisms, the glycosyltransferase 635 00:32:28,360 --> 00:32:31,260 and the transpeptidase are on the same protein. 636 00:32:31,260 --> 00:32:32,800 They're two domains. 637 00:32:32,800 --> 00:32:37,320 But, in many organisms, they're not. 638 00:32:37,320 --> 00:32:42,340 OK, so you have two separate proteins. 639 00:32:42,340 --> 00:32:44,320 And furthermore, in Staph. aureus 640 00:32:44,320 --> 00:32:47,360 there are now five of these kinds of proteins. 641 00:32:47,360 --> 00:32:49,060 So the question is, what are all five 642 00:32:49,060 --> 00:32:51,460 of these glycosyltransferase doing? 643 00:32:51,460 --> 00:32:52,960 Which ones are involved in which? 644 00:32:52,960 --> 00:32:56,853 Which ones are involved in antibiotic resistance? 645 00:32:56,853 --> 00:32:59,020 And I think, when you start looking at it like this, 646 00:32:59,020 --> 00:33:00,550 you know, it's very complex. 647 00:33:00,550 --> 00:33:04,420 You realize what a hard problem this actually is. 648 00:33:04,420 --> 00:33:05,980 But we now have the tools, I think, 649 00:33:05,980 --> 00:33:07,360 because of beautiful studies that 650 00:33:07,360 --> 00:33:09,160 have been done in the last few years, 651 00:33:09,160 --> 00:33:10,660 to start investigating this. 652 00:33:10,660 --> 00:33:15,160 So this just shows, here, again, we have our lipid 2. 653 00:33:15,160 --> 00:33:16,930 We have our growing chain. 654 00:33:16,930 --> 00:33:18,740 And here we have our pentaglycine. 655 00:33:18,740 --> 00:33:21,550 So this is Staph. aureus. 656 00:33:21,550 --> 00:33:27,760 And we take D-alinine D-alinine, and form a cross-link 657 00:33:27,760 --> 00:33:29,740 and kick out D-alinine. 658 00:33:29,740 --> 00:33:31,720 And many of you have probably seen this before. 659 00:33:31,720 --> 00:33:34,070 I used to teach this in high school. 660 00:33:34,070 --> 00:33:36,820 [LAUGH] So that D-alinine D-alinine 661 00:33:36,820 --> 00:33:38,710 looks like penicillin. 662 00:33:38,710 --> 00:33:41,140 And we understand that this works-- 663 00:33:41,140 --> 00:33:43,580 it looks amazing, like a serine protease, which 664 00:33:43,580 --> 00:33:44,920 you're all very familiar with. 665 00:33:44,920 --> 00:33:47,140 We've seen this hundreds of times, now, 666 00:33:47,140 --> 00:33:48,250 in the earlier part-- 667 00:33:48,250 --> 00:33:49,900 to form this cross-link. 668 00:33:49,900 --> 00:33:53,890 And that cross-link is essential for the viability 669 00:33:53,890 --> 00:33:56,590 of the organism, in different ways. 670 00:33:56,590 --> 00:33:59,800 And you can imagine, if a bacteria is dividing, 671 00:33:59,800 --> 00:34:02,620 that you might have different peptidoglycal structure 672 00:34:02,620 --> 00:34:06,010 at the site, where the two dividing bacteria are 673 00:34:06,010 --> 00:34:07,390 going to split apart. 674 00:34:07,390 --> 00:34:08,949 So that might be why you want to have 675 00:34:08,949 --> 00:34:14,210 multiple glycosyltransferases in this overall process. 676 00:34:14,210 --> 00:34:14,710 OK. 677 00:34:14,710 --> 00:34:19,909 So this is just a cartoon that shows you targets. 678 00:34:19,909 --> 00:34:22,219 These are all natural products. 679 00:34:22,219 --> 00:34:23,710 Here's penicillin. 680 00:34:23,710 --> 00:34:24,460 It targets-- 681 00:34:24,460 --> 00:34:26,830 It looks-- not in this picture, but you 682 00:34:26,830 --> 00:34:28,030 can use your imagination. 683 00:34:28,030 --> 00:34:31,360 It looks just like D-alinine D-alinine. 684 00:34:31,360 --> 00:34:34,239 Binds in the active site, and covalently 685 00:34:34,239 --> 00:34:37,570 modifies a serine involved in that reaction. 686 00:34:37,570 --> 00:34:38,440 Moenomycin. 687 00:34:38,440 --> 00:34:40,150 What does this look like? 688 00:34:40,150 --> 00:34:41,260 This is sort of amazing. 689 00:34:41,260 --> 00:34:43,600 It's got this lipid thing, hanging off the end. 690 00:34:43,600 --> 00:34:45,159 That's a natural product. 691 00:34:45,159 --> 00:34:49,480 It binds, also, to the glycosyltransferase. 692 00:34:49,480 --> 00:34:51,730 And people are actively investigating this. 693 00:34:51,730 --> 00:34:54,340 You can imagine, this is not so easy to make 694 00:34:54,340 --> 00:34:57,030 as a new antibiotic. 695 00:34:57,030 --> 00:35:00,010 And then we have vancomycin, and vancomycin 696 00:35:00,010 --> 00:35:02,340 is able to bind D-alinine D-alinine. 697 00:35:02,340 --> 00:35:06,160 So these are all natural products that target cell wall. 698 00:35:06,160 --> 00:35:09,450 And, by far and away, the penicillins 699 00:35:09,450 --> 00:35:11,840 are the ones that are used much more prevalently. 700 00:35:11,840 --> 00:35:13,810 We have hundreds of variations of the theme. 701 00:35:13,810 --> 00:35:17,680 And, again, it's the war between the bacteria and the human, 702 00:35:17,680 --> 00:35:20,860 to figure out how to keep themselves growing. 703 00:35:20,860 --> 00:35:24,190 And so we have many variations on the beta-lactams. 704 00:35:24,190 --> 00:35:27,420 And you can take this even a step further, if you go-- 705 00:35:27,420 --> 00:35:30,550 in addition to the peptidoglycan, 706 00:35:30,550 --> 00:35:34,030 you have polymers of teichoic acid-- 707 00:35:34,030 --> 00:35:35,680 which I'm not [LAUGH] going to go into. 708 00:35:35,680 --> 00:35:38,820 But now people, for the first time, this year, 709 00:35:38,820 --> 00:35:42,040 have been able to reconstitute this polymer biosynthetic 710 00:35:42,040 --> 00:35:42,610 pathway. 711 00:35:42,610 --> 00:35:46,570 And this is a new target for design of the antibacterial. 712 00:35:46,570 --> 00:35:49,540 So I think it's exciting times, and we have really smart people 713 00:35:49,540 --> 00:35:51,520 working on this problem. 714 00:35:51,520 --> 00:35:54,310 And they now, for the first time, can set up the assays, 715 00:35:54,310 --> 00:35:56,350 so they can screen for small molecules that 716 00:35:56,350 --> 00:36:00,400 hopefully can target cell wall, which is unique to bacteria. 717 00:36:00,400 --> 00:36:01,270 OK. 718 00:36:01,270 --> 00:36:04,540 So what I want to do is talk about, 719 00:36:04,540 --> 00:36:06,100 in the last few minutes, as we're now 720 00:36:06,100 --> 00:36:07,460 moving into Staph. aureus. 721 00:36:07,460 --> 00:36:07,960 OK? 722 00:36:07,960 --> 00:36:10,690 And we're going to focus in on heme uptake rather 723 00:36:10,690 --> 00:36:12,130 than siderophore uptake. 724 00:36:12,130 --> 00:36:15,940 But if you look at this, what do we know about Staph. aureus? 725 00:36:15,940 --> 00:36:20,080 We know what a bit, because everybody and his brother 726 00:36:20,080 --> 00:36:22,810 has been studying it because of the problems with resistance. 727 00:36:22,810 --> 00:36:28,210 So, here, again, Staph. aureus actually 728 00:36:28,210 --> 00:36:34,190 has two biosynthetic pathways encoded in its genome. 729 00:36:34,190 --> 00:36:39,590 And what these pathways code for are these two siderophores. 730 00:36:39,590 --> 00:36:40,090 OK? 731 00:36:40,090 --> 00:36:42,830 And if you look at this, what's unusual? 732 00:36:42,830 --> 00:36:44,440 Does anybody see anything unusual 733 00:36:44,440 --> 00:36:47,680 about the siderophore structure, if you look at it carefully? 734 00:36:51,340 --> 00:36:53,550 I don't want to spend a lot of time on this, 735 00:36:53,550 --> 00:36:55,690 but what do you see in the structure? 736 00:36:55,690 --> 00:36:56,600 Can you read it? 737 00:36:56,600 --> 00:36:59,090 Or, if you brought your handout, you can probably read it. 738 00:36:59,090 --> 00:37:01,370 Since I insist on having the windows open, 739 00:37:01,370 --> 00:37:02,610 it's harder to read this. 740 00:37:02,610 --> 00:37:06,800 But what do we see, in siderophore, 741 00:37:06,800 --> 00:37:11,840 in this siderophore, Staphyloferrin A? 742 00:37:11,840 --> 00:37:13,960 See anything you recognize? 743 00:37:13,960 --> 00:37:14,460 Yeah. 744 00:37:14,460 --> 00:37:15,260 AUDIENCE: Some citrates? 745 00:37:15,260 --> 00:37:16,510 JOANNE STUBBE: Yeah, citrates. 746 00:37:16,510 --> 00:37:18,410 So, again, we're using citrate. 747 00:37:18,410 --> 00:37:22,400 We saw polycitrate can bind iron as a siderophore in itself. 748 00:37:22,400 --> 00:37:24,170 And, in fact, most gram-negative bacteria 749 00:37:24,170 --> 00:37:27,310 have iron-siderophore uptake system. 750 00:37:27,310 --> 00:37:29,280 Here, actually, all of these-- 751 00:37:29,280 --> 00:37:32,720 if you look at this carefully, the biosynthetic pathway, 752 00:37:32,720 --> 00:37:36,060 you know, is made out of basic metabolites. 753 00:37:36,060 --> 00:37:36,560 OK? 754 00:37:36,560 --> 00:37:40,190 That you see out of normal, central metabolic pathways. 755 00:37:40,190 --> 00:37:46,430 And what happens is, there's an ABC transporter and an ATPase-- 756 00:37:46,430 --> 00:37:48,270 FhuC is an ATPase-- 757 00:37:48,270 --> 00:37:50,900 all of this is written down in your notes-- 758 00:37:50,900 --> 00:37:57,080 that allow the siderophore to bring iron into the cell. 759 00:37:57,080 --> 00:37:58,580 And I think what's interesting here, 760 00:37:58,580 --> 00:38:00,500 and I've already pointed this out, 761 00:38:00,500 --> 00:38:05,880 in addition to the siderophores that the organism makes it also 762 00:38:05,880 --> 00:38:12,750 has a generic transporter that allows siderophores 763 00:38:12,750 --> 00:38:16,000 made by other organisms to bring iron into the cell. 764 00:38:16,000 --> 00:38:18,900 And so, again, that's a strategy that's 765 00:38:18,900 --> 00:38:20,380 used over and over again. 766 00:38:20,380 --> 00:38:25,410 So here's a xenosiderophore transport system, 767 00:38:25,410 --> 00:38:27,070 desperately trying to get iron. 768 00:38:27,070 --> 00:38:27,690 OK. 769 00:38:27,690 --> 00:38:30,690 So the ones we're going to be talking about and focusing on 770 00:38:30,690 --> 00:38:34,158 specifically are the heme uptake systems. 771 00:38:34,158 --> 00:38:35,700 And these are the ones you've already 772 00:38:35,700 --> 00:38:39,180 hopefully thought about, now, from your problem set. 773 00:38:39,180 --> 00:38:41,040 We have to extract-- 774 00:38:41,040 --> 00:38:45,670 I just told you that red blood cells have most of the iron. 775 00:38:45,670 --> 00:38:47,010 So Staph. 776 00:38:47,010 --> 00:38:50,820 has been incredibly creative in generating endotoxins 777 00:38:50,820 --> 00:38:54,870 that lyse red blood cells, allowing the heme-- 778 00:38:54,870 --> 00:38:56,640 hemoglobin, OK? 779 00:38:56,640 --> 00:39:07,320 So we have endotoxins from the organism 780 00:39:07,320 --> 00:39:12,070 that lyse red blood cells. 781 00:39:12,070 --> 00:39:16,330 And so what you get out, then, is hemoglobin. 782 00:39:16,330 --> 00:39:19,960 Which, again, has four hemes and iron. 783 00:39:19,960 --> 00:39:21,250 And you want to get-- 784 00:39:21,250 --> 00:39:23,500 the key thing is to get the iron out of the heme. 785 00:39:23,500 --> 00:39:27,220 So you want to be able to extract the iron out 786 00:39:27,220 --> 00:39:27,820 of the heme. 787 00:39:27,820 --> 00:39:32,255 And also-- and I have this down in your nomenclature-- 788 00:39:32,255 --> 00:39:34,630 it turns out red blood cells have another protein, called 789 00:39:34,630 --> 00:39:37,150 "haptoglobin," that binds to hemoglobin. 790 00:39:37,150 --> 00:39:39,820 And that's another place that these organisms have 791 00:39:39,820 --> 00:39:42,190 evolved to extract the heme-- 792 00:39:42,190 --> 00:39:44,170 to extract the heme. 793 00:39:44,170 --> 00:39:45,670 So, in all of these cases, you're 794 00:39:45,670 --> 00:39:52,270 extracting the heme out of the protein. 795 00:39:52,270 --> 00:39:57,760 And so, over here, you see the two different ways to do that. 796 00:39:57,760 --> 00:40:01,040 And we have different proteins that are able to do this. 797 00:40:01,040 --> 00:40:04,210 And then, eventually, the heme that's extracted 798 00:40:04,210 --> 00:40:06,970 is passed through this peptidoglycan, 799 00:40:06,970 --> 00:40:11,440 eventually to the plasma membrane, where 800 00:40:11,440 --> 00:40:13,510 the heme goes into the cytosol. 801 00:40:13,510 --> 00:40:15,590 And in this organism, to get it out, 802 00:40:15,590 --> 00:40:17,110 you have to break down the heme. 803 00:40:17,110 --> 00:40:21,730 You have to cleave it into pieces by the enzyme called 804 00:40:21,730 --> 00:40:23,340 "heme oxygenases." 805 00:40:23,340 --> 00:40:24,280 OK. 806 00:40:24,280 --> 00:40:26,830 So I don't want to really say very much 807 00:40:26,830 --> 00:40:32,290 about the siderophores, except to say-- 808 00:40:32,290 --> 00:40:35,950 let me comment on iron sensing. 809 00:40:35,950 --> 00:40:40,570 And you saw-- and this would be Staph. aureus, 810 00:40:40,570 --> 00:40:45,640 but it's in true iron-sensing for most bacteria. 811 00:40:45,640 --> 00:40:47,950 You saw iron-sensing predominantly 812 00:40:47,950 --> 00:40:49,450 at the translational level. 813 00:40:49,450 --> 00:40:50,430 Which was unusual. 814 00:40:50,430 --> 00:40:52,450 That's why we talked about it, in humans. 815 00:40:52,450 --> 00:40:55,330 Here, iron-sensing is predominantly 816 00:40:55,330 --> 00:40:57,130 at the transcriptional level. 817 00:40:57,130 --> 00:41:00,770 So this sensing occurs transcriptionally. 818 00:41:05,870 --> 00:41:12,640 And so you have a transcription factor, which is called "Fur." 819 00:41:12,640 --> 00:41:14,350 And Fur is a transcription factor. 820 00:41:14,350 --> 00:41:18,400 That name is used for almost all organisms. 821 00:41:18,400 --> 00:41:21,760 And I'm not going to say much about this, 822 00:41:21,760 --> 00:41:25,460 but we're going to look at the operon in a minute. 823 00:41:25,460 --> 00:41:28,840 But here's Fur. 824 00:41:28,840 --> 00:41:36,870 And if Fur has iron bound, what it does is a repressor. 825 00:41:36,870 --> 00:41:42,340 And it shuts down transcription of all the proteins 826 00:41:42,340 --> 00:41:44,560 that you might think it would shut down. 827 00:41:44,560 --> 00:41:47,920 They can no longer take up iron into the cell, 828 00:41:47,920 --> 00:41:50,420 because you have excess iron and you don't need anymore. 829 00:41:50,420 --> 00:41:52,900 Again, you want to control iron, because you 830 00:41:52,900 --> 00:41:55,210 have problems if you have too much iron 831 00:41:55,210 --> 00:41:57,130 with oxidative stress. 832 00:41:57,130 --> 00:41:57,630 OK. 833 00:41:57,630 --> 00:42:00,530 So, if you look at the operon-- 834 00:42:00,530 --> 00:42:01,030 let's see. 835 00:42:01,030 --> 00:42:04,650 So look at the operon, here. 836 00:42:04,650 --> 00:42:06,110 So here's the operon. 837 00:42:06,110 --> 00:42:08,860 And we're going to see that the key proteins involved 838 00:42:08,860 --> 00:42:13,010 in heme uptake are called the "Isd" proteins. 839 00:42:13,010 --> 00:42:18,470 And so, if you look at all of these Isd proteins, this Isd 840 00:42:18,470 --> 00:42:22,310 protein and that one, they all have these little Fur boxes. 841 00:42:22,310 --> 00:42:25,370 [LAUGH] So we have a Fur box ahead, 842 00:42:25,370 --> 00:42:28,700 which regulates whether you're going to make a siderophore 843 00:42:28,700 --> 00:42:31,640 or whether you're going to make all this equipment required 844 00:42:31,640 --> 00:42:32,780 to take up heme. 845 00:42:32,780 --> 00:42:35,420 So all of that makes sense, and people 846 00:42:35,420 --> 00:42:40,310 have studied this extensively, in many of these organisms. 847 00:42:40,310 --> 00:42:40,970 OK. 848 00:42:40,970 --> 00:42:45,770 So what I want to do now is, I'm going to show you this cartoon 849 00:42:45,770 --> 00:42:46,770 overview. 850 00:42:46,770 --> 00:42:49,790 And then we'll look at a few experiments 851 00:42:49,790 --> 00:42:56,030 that people have done to try to look at what basis in reality 852 00:42:56,030 --> 00:42:58,850 this cartoon model has to what actually happens 853 00:42:58,850 --> 00:43:00,200 inside the cell. 854 00:43:00,200 --> 00:43:01,632 So let's look at-- 855 00:43:01,632 --> 00:43:03,590 I can never remember the names of these things. 856 00:43:03,590 --> 00:43:07,670 I'm just going to call it the "Isd proteins." 857 00:43:07,670 --> 00:43:10,340 And so there are two proteins, we're going to see, 858 00:43:10,340 --> 00:43:14,540 that are closest to the surface, that directly interact with 859 00:43:14,540 --> 00:43:17,180 hemoglobin-- or haptoglobin and hemoglobin-- 860 00:43:17,180 --> 00:43:19,400 the other ones that are going to somehow get 861 00:43:19,400 --> 00:43:22,700 the heme out of the proteins. 862 00:43:22,700 --> 00:43:27,100 And then these each have little NEAT domains. 863 00:43:27,100 --> 00:43:29,630 So N1 is a NEAT domain. 864 00:43:29,630 --> 00:43:32,840 So they have a name for that, which I've also written down. 865 00:43:32,840 --> 00:43:35,120 It's, like, 120 amino acids. 866 00:43:35,120 --> 00:43:37,880 And each one of these proteins sometimes has two, 867 00:43:37,880 --> 00:43:40,100 sometimes has three, sometimes has one, 868 00:43:40,100 --> 00:43:42,480 and they're structurally all the same. 869 00:43:42,480 --> 00:43:46,940 But it turns out that you can't just pick up one and replace it 870 00:43:46,940 --> 00:43:47,900 with another. 871 00:43:47,900 --> 00:43:49,400 There's something about the spinach 872 00:43:49,400 --> 00:43:51,860 on each side of these NEAT domains 873 00:43:51,860 --> 00:43:56,600 that is key, you can imagine, for the directionality 874 00:43:56,600 --> 00:43:57,740 of the transfer. 875 00:43:57,740 --> 00:44:02,690 So you want something that the heme is going to get down here. 876 00:44:02,690 --> 00:44:04,790 You don't want something where the equilibrium 877 00:44:04,790 --> 00:44:06,470 is going to stay up there. 878 00:44:06,470 --> 00:44:08,610 So this is not an easy problem. 879 00:44:08,610 --> 00:44:12,650 And this is a problem that we discussed in the beginning-- 880 00:44:12,650 --> 00:44:15,770 the importance of exchange ligands. 881 00:44:15,770 --> 00:44:18,560 Because somehow we're going to have a heme in a little NEAT 882 00:44:18,560 --> 00:44:21,500 domain, but it's going to move into the next domain. 883 00:44:21,500 --> 00:44:22,850 It just doesn't hop. 884 00:44:22,850 --> 00:44:24,620 It's covalently bound. 885 00:44:24,620 --> 00:44:28,490 So how do you transfer one heme to the next heme? 886 00:44:28,490 --> 00:44:30,470 And we have a lot of structural information, 887 00:44:30,470 --> 00:44:32,900 but I would say we still don't understand how 888 00:44:32,900 --> 00:44:35,180 these transfers actually occur. 889 00:44:35,180 --> 00:44:35,810 OK. 890 00:44:35,810 --> 00:44:38,477 So there's a couple other things that I want to point out, here. 891 00:44:38,477 --> 00:44:44,600 So IsB and IsH extract from heme and hemoglobin. 892 00:44:44,600 --> 00:44:47,000 This gives you a feeling, which you also 893 00:44:47,000 --> 00:44:50,540 saw from the problem set, that these little domains-- 894 00:44:50,540 --> 00:44:53,060 N1 domains, N2 domains-- 895 00:44:53,060 --> 00:44:54,320 are all NEAT domains. 896 00:44:54,320 --> 00:44:56,798 So we have multiple domains. 897 00:44:56,798 --> 00:44:58,340 And what we're going to see, and this 898 00:44:58,340 --> 00:45:01,790 is key to the way these organisms function, 899 00:45:01,790 --> 00:45:05,750 is that these Isd proteins are covalently 900 00:45:05,750 --> 00:45:07,790 attached to the peptidoglycan. 901 00:45:07,790 --> 00:45:21,630 So the issue is, we need to covalently attach the Isd 902 00:45:21,630 --> 00:45:28,800 proteins to the peptidoglycans. 903 00:45:28,800 --> 00:45:31,890 And the protein-- 904 00:45:31,890 --> 00:45:35,280 There are two different proteins that do this. 905 00:45:35,280 --> 00:45:43,410 So the Isd proteins have ZIP codes. 906 00:45:43,410 --> 00:45:44,520 Where have we seen this? 907 00:45:44,520 --> 00:45:48,060 We see this over and over and over again. 908 00:45:48,060 --> 00:45:50,820 We have little sequences of peptides that are 909 00:45:50,820 --> 00:45:53,220 recognized by another protein. 910 00:45:53,220 --> 00:45:54,380 OK? 911 00:45:54,380 --> 00:45:55,560 So we have ZIP codes. 912 00:45:55,560 --> 00:45:57,990 And the ZIP codes, I'll just say "see PowerPoint" 913 00:45:57,990 --> 00:46:00,420 for the sequence. 914 00:46:00,420 --> 00:46:04,110 And it turns out, if you look over here, 915 00:46:04,110 --> 00:46:07,530 all of these proteins with a yellow anchor 916 00:46:07,530 --> 00:46:08,950 have little ZIP codes in them. 917 00:46:08,950 --> 00:46:09,450 OK? 918 00:46:09,450 --> 00:46:11,760 [LAUGH] And they're recognized by a protein 919 00:46:11,760 --> 00:46:13,560 called "sortase A." 920 00:46:13,560 --> 00:46:14,060 OK. 921 00:46:14,060 --> 00:46:18,712 So we'll see that, in addition to the Ist proteins, 922 00:46:18,712 --> 00:46:19,420 we have sortases. 923 00:46:22,590 --> 00:46:26,760 And we have sortase A and B, and they 924 00:46:26,760 --> 00:46:35,360 recognize the ZIP codes, distinct ZIP codes, 925 00:46:35,360 --> 00:46:39,540 and are required to attach the Isd proteins covalently 926 00:46:39,540 --> 00:46:41,010 to the peptidoglycan. 927 00:46:41,010 --> 00:46:47,970 And in the peptidoglycan of any gram-positive, a lot of things 928 00:46:47,970 --> 00:46:51,270 are covalently attached to the peptidoglycan. 929 00:46:51,270 --> 00:46:52,890 So, I mean, can you imagine-- how 930 00:46:52,890 --> 00:46:56,790 dense do you need these proteins, to be 931 00:46:56,790 --> 00:46:58,110 able to do these switches? 932 00:46:58,110 --> 00:47:00,270 I mean, this is a cartoon overview 933 00:47:00,270 --> 00:47:02,850 that really doesn't tell you anything 934 00:47:02,850 --> 00:47:04,320 about the complexity of all that-- 935 00:47:04,320 --> 00:47:06,190 what does a peptidoglycan look like? 936 00:47:06,190 --> 00:47:09,780 Well, it's got a lot of water and a lot of space 937 00:47:09,780 --> 00:47:12,100 in between these N-acetylglucosamine, 938 00:47:12,100 --> 00:47:14,410 N-acetylmuramic acids. 939 00:47:14,410 --> 00:47:18,080 So this is involved in the covalent attachment. 940 00:47:21,920 --> 00:47:25,640 And it, in fact, involves what you've seen over 941 00:47:25,640 --> 00:47:31,770 and over again-- involves covalent catalysis 942 00:47:31,770 --> 00:47:35,160 with a cystine in its active site. 943 00:47:35,160 --> 00:47:36,240 OK? 944 00:47:36,240 --> 00:47:41,670 So what I want to do is briefly look at what 945 00:47:41,670 --> 00:47:43,325 these sortases actually do. 946 00:47:43,325 --> 00:47:44,950 I'm not going to write it on the board. 947 00:47:44,950 --> 00:47:48,755 I'll walk you through it and then, next time-- 948 00:47:48,755 --> 00:47:50,130 hopefully, you've already thought 949 00:47:50,130 --> 00:47:51,840 about this in some form, but I'll walk you through it 950 00:47:51,840 --> 00:47:53,462 and go through it next time. 951 00:47:53,462 --> 00:47:55,170 And then what we're going to do is simply 952 00:47:55,170 --> 00:47:58,260 look at a few experiments with Isd proteins, 953 00:47:58,260 --> 00:48:01,740 to look at this movement of heme across the membrane, 954 00:48:01,740 --> 00:48:03,870 similar to the kinds of experiments 955 00:48:03,870 --> 00:48:08,700 that you had on the problem set that was due this week. 956 00:48:08,700 --> 00:48:09,230 OK. 957 00:48:09,230 --> 00:48:11,280 So, because I don't have much time 958 00:48:11,280 --> 00:48:13,968 and I can't write that fast and you can't write that fast, 959 00:48:13,968 --> 00:48:15,510 either, [LAUGH] I'm going to walk you 960 00:48:15,510 --> 00:48:18,150 through sort of what's going on in this reaction. 961 00:48:18,150 --> 00:48:18,870 OK. 962 00:48:18,870 --> 00:48:21,360 So, remember, all of these things 963 00:48:21,360 --> 00:48:24,370 are anchored to the plasma membrane. 964 00:48:24,370 --> 00:48:25,620 OK, so that's the other thing. 965 00:48:25,620 --> 00:48:28,740 Sometimes they have single, transmembrane-spanning regions. 966 00:48:28,740 --> 00:48:31,560 Sometimes they have lipids that are actually bound. 967 00:48:31,560 --> 00:48:33,850 I wanted to say one other thing, here. 968 00:48:33,850 --> 00:48:36,750 So these yellow things are anchored 969 00:48:36,750 --> 00:48:41,100 by sortase A. The blue thing is anchored by sortase B. 970 00:48:41,100 --> 00:48:45,870 And IsdE is anchored by a lipid, covalently bound. 971 00:48:45,870 --> 00:48:48,990 OK, so we have three different strategies, to anchor. 972 00:48:48,990 --> 00:48:50,220 OK? 973 00:48:50,220 --> 00:48:52,630 And every organism is distinct. 974 00:48:52,630 --> 00:48:55,200 Whoops, I'm going the wrong way. 975 00:48:55,200 --> 00:48:57,550 OK, so what happens in this reaction? 976 00:48:57,550 --> 00:49:00,170 So here's our ZIP code. 977 00:49:00,170 --> 00:49:03,810 OK, and what we know about this-- and here's sortase A. 978 00:49:03,810 --> 00:49:08,030 Sortase A is anchored to the plasma membrane. 979 00:49:08,030 --> 00:49:11,060 In a further cartoon, they don't have it anchored, 980 00:49:11,060 --> 00:49:13,610 but I tell you it's anchored. 981 00:49:13,610 --> 00:49:17,960 And we know we get cleavage between threonine and glycine. 982 00:49:17,960 --> 00:49:20,450 And we know we have a sulfhydryl on the active site. 983 00:49:20,450 --> 00:49:24,290 So, this chemistry, we've seen over and over and over and over 984 00:49:24,290 --> 00:49:26,840 again, whether it's with serine or with a cystine, 985 00:49:26,840 --> 00:49:28,640 you have to have the right equipment 986 00:49:28,640 --> 00:49:30,930 to acylate the enzyme. 987 00:49:30,930 --> 00:49:34,700 So what happens here is you acylate the enzyme. 988 00:49:34,700 --> 00:49:38,120 And so this is the part of the protein that's 989 00:49:38,120 --> 00:49:42,240 going to get transferred, ultimately, to-- this 990 00:49:42,240 --> 00:49:45,390 is lipid 2, with the pentaglycine. 991 00:49:45,390 --> 00:49:47,100 And at the end of the pentaglycine 992 00:49:47,100 --> 00:49:48,810 you have an amino group. 993 00:49:48,810 --> 00:49:51,600 That's-- you're going to attach this protein, 994 00:49:51,600 --> 00:49:57,650 IsdA or IsdB to this lyse-- 995 00:49:57,650 --> 00:50:05,520 the end-terminal amino group of glycine in the pentaglycine. 996 00:50:05,520 --> 00:50:06,900 So you form. 997 00:50:06,900 --> 00:50:11,040 Again, you cleave this peptide bond. 998 00:50:11,040 --> 00:50:15,630 And you have this piece left over from your Isd protein. 999 00:50:15,630 --> 00:50:18,900 You now have this covalently attached to the sortase. 1000 00:50:18,900 --> 00:50:21,000 And again, what you're doing is going 1001 00:50:21,000 --> 00:50:23,280 to regenerate the sortase, so you 1002 00:50:23,280 --> 00:50:25,330 can do more of these reactions. 1003 00:50:25,330 --> 00:50:31,640 And here you're forming your linkage to the Isd protein. 1004 00:50:31,640 --> 00:50:32,140 OK? 1005 00:50:32,140 --> 00:50:34,840 Does everybody see what's going on in that reaction? 1006 00:50:34,840 --> 00:50:38,760 So another cartoon version of this, and then I'll stop here. 1007 00:50:38,760 --> 00:50:40,290 This is a more chemical version. 1008 00:50:40,290 --> 00:50:43,560 Again, this is the sortase. 1009 00:50:43,560 --> 00:50:45,320 Here is your amino-acid sequence. 1010 00:50:45,320 --> 00:50:47,070 You go through a tetrahedral intermediate. 1011 00:50:47,070 --> 00:50:49,545 This is all a figment of our imaginations, 1012 00:50:49,545 --> 00:50:53,250 [LAUGH] based on what we think-- what we do understand, 1013 00:50:53,250 --> 00:50:56,880 in the test tube of peptide bond hydrolysis-- 1014 00:50:56,880 --> 00:50:59,220 not so much in the enzymes. 1015 00:50:59,220 --> 00:51:04,530 But you generate an acylated attached protein. 1016 00:51:04,530 --> 00:51:06,780 And then we have our pentaglycine, 1017 00:51:06,780 --> 00:51:09,960 the terminal amino group that goes through, 1018 00:51:09,960 --> 00:51:12,840 again, a tetrahedral intermediate to form 1019 00:51:12,840 --> 00:51:14,820 this linkage. 1020 00:51:14,820 --> 00:51:17,670 So what's happening-- I think this is, like, so, again, 1021 00:51:17,670 --> 00:51:18,180 amazing-- 1022 00:51:18,180 --> 00:51:21,720 what's happening is, you're transferring-- 1023 00:51:21,720 --> 00:51:24,360 you've got your lipid 2, and you've 1024 00:51:24,360 --> 00:51:27,450 transferred it across this membrane 1025 00:51:27,450 --> 00:51:31,120 into the outside of your bacteria. 1026 00:51:31,120 --> 00:51:32,370 So you've gotta hang it there. 1027 00:51:32,370 --> 00:51:35,670 That's why you need these big, huge lipids. 1028 00:51:35,670 --> 00:51:39,740 And what you're going to do is attach to this pentaglycine. 1029 00:51:39,740 --> 00:51:45,540 You're going to attach each of these Isd proteins, covalently. 1030 00:51:45,540 --> 00:51:46,850 And then what you do-- 1031 00:51:46,850 --> 00:51:48,000 So you make this guy. 1032 00:51:48,000 --> 00:51:52,090 Then you attach this whole thing onto the growing polypeptide 1033 00:51:52,090 --> 00:51:52,590 chain. 1034 00:51:52,590 --> 00:51:55,020 I mean, this is, like, an amazing machine 1035 00:51:55,020 --> 00:51:57,900 that they've unraveled, I think, from studies 1036 00:51:57,900 --> 00:52:01,600 that have been done in the last five years or so. 1037 00:52:01,600 --> 00:52:04,650 So, next time, we'll come back and talk a little bit 1038 00:52:04,650 --> 00:52:06,960 about the Isd proteins, but I think 1039 00:52:06,960 --> 00:52:08,200 you should be fine, looking. 1040 00:52:08,200 --> 00:52:10,575 You've looked at-- all you're doing is transferring heme, 1041 00:52:10,575 --> 00:52:12,660 and we don't understand the detailed mechanism 1042 00:52:12,660 --> 00:52:13,890 of how that happens. 1043 00:52:13,890 --> 00:52:17,510 That's something hopefully some of you will figure out.