1 00:00:00,500 --> 00:00:02,810 The following content is provided under a Creative 2 00:00:02,810 --> 00:00:04,380 Commons license. 3 00:00:04,380 --> 00:00:06,670 Your support will help MIT OpenCourseWare 4 00:00:06,670 --> 00:00:11,010 continue to offer high quality educational resources for free. 5 00:00:11,010 --> 00:00:13,670 To make a donation or view additional materials 6 00:00:13,670 --> 00:00:17,600 from hundreds of MIT courses, visit MIT OpenCourseWare 7 00:00:17,600 --> 00:00:18,460 at ocw.mit.edu. 8 00:00:25,494 --> 00:00:27,760 ELIZABETH NOLAN: --by talking about ClpX. 9 00:00:27,760 --> 00:00:31,720 And then we're going to move into module 4-- 10 00:00:31,720 --> 00:00:34,930 which is the last module before spring break-- 11 00:00:34,930 --> 00:00:38,800 on synthases and assembly line biosynthesis. 12 00:00:44,690 --> 00:00:51,640 So basically last time, where we left off is, 13 00:00:51,640 --> 00:00:53,560 we went over experiments that were 14 00:00:53,560 --> 00:00:56,920 done to look at denaturation, translocation, 15 00:00:56,920 --> 00:00:59,910 and degradation by ClpXP. 16 00:00:59,910 --> 00:01:04,390 And closed with a question about what actually is going on 17 00:01:04,390 --> 00:01:07,930 in ClpX with this ATP binding and hydrolysis 18 00:01:07,930 --> 00:01:11,350 to allow for these condemned protein 19 00:01:11,350 --> 00:01:14,410 substrates to be unfolded and translocated 20 00:01:14,410 --> 00:01:16,900 into the degradation chamber. 21 00:01:16,900 --> 00:01:23,140 And I left you just with the statement that although we 22 00:01:23,140 --> 00:01:37,800 think about ClpX as this hexamer that has six identical 23 00:01:37,800 --> 00:01:41,160 subunits, what studies have shown is that there's some 24 00:01:41,160 --> 00:01:44,988 inherent asymmetry within this AAA+ ATPase. 25 00:01:44,988 --> 00:01:47,280 And that's what we're going to talk about a little bit. 26 00:01:47,280 --> 00:01:50,550 So this is just a slide from a few lectures ago 27 00:01:50,550 --> 00:01:53,880 that's showing the top view and side view of ClpX 28 00:01:53,880 --> 00:01:55,260 and how we've thought about this. 29 00:01:55,260 --> 00:01:58,470 And I think these studies just really 30 00:01:58,470 --> 00:02:02,940 highlight how complicated these machines are 31 00:02:02,940 --> 00:02:05,100 and that there's still a lot more we 32 00:02:05,100 --> 00:02:06,780 need to figure out here. 33 00:02:06,780 --> 00:02:10,860 So as I said last time, this asymmetry 34 00:02:10,860 --> 00:02:15,330 comes from whether or not each ClpX subunit 35 00:02:15,330 --> 00:02:17,460 is bound to nucleotide. 36 00:02:17,460 --> 00:02:22,440 And so basically, from looking at many different crystal 37 00:02:22,440 --> 00:02:35,410 structures, what can be done is that the ClpX subunits 38 00:02:35,410 --> 00:02:43,960 can be divided into two different types based 39 00:02:43,960 --> 00:02:51,950 on conformation here. 40 00:02:51,950 --> 00:02:53,570 And so in thinking about this, we 41 00:02:53,570 --> 00:02:58,730 want to first think about the ClpX domain organization. 42 00:02:58,730 --> 00:03:02,240 And if we just think about this, what ClpX has is 43 00:03:02,240 --> 00:03:05,930 an end domain followed by a domain 44 00:03:05,930 --> 00:03:11,660 that's called the large domain and then followed 45 00:03:11,660 --> 00:03:12,905 by a small domain. 46 00:03:18,050 --> 00:03:22,520 So 633 amino acids, just to give you a sense of size, 47 00:03:22,520 --> 00:03:27,430 and about 69 kilodaltons per subunit. 48 00:03:27,430 --> 00:03:29,180 And so what we're going to focus on 49 00:03:29,180 --> 00:03:31,880 are the large and the small subunits 50 00:03:31,880 --> 00:03:35,930 and what's observed from many different crystal structures. 51 00:03:35,930 --> 00:03:40,130 And so these two different types of subunit 52 00:03:40,130 --> 00:03:43,940 have been described as loadable and unloadable, 53 00:03:43,940 --> 00:03:48,100 and that depends on whether or not nucleotide is bound. 54 00:03:48,100 --> 00:03:51,890 So if we consider of these two types, 55 00:03:51,890 --> 00:03:55,040 just thinking about the large and small domains, 56 00:03:55,040 --> 00:04:01,000 we have this loadable arrangement 57 00:04:01,000 --> 00:04:02,470 which binds nucleotide. 58 00:04:08,340 --> 00:04:11,245 And in cartoon, something like this. 59 00:04:11,245 --> 00:04:15,626 So we have the large subunit. 60 00:04:15,626 --> 00:04:16,709 We have the small subunit. 61 00:04:20,310 --> 00:04:22,304 And we have this region that's called a hinge. 62 00:04:26,520 --> 00:04:28,260 So this is one ClpX. 63 00:04:33,915 --> 00:04:35,060 So ATP binds. 64 00:04:38,140 --> 00:04:42,080 And so the other type is described as unloadable. 65 00:04:48,390 --> 00:04:54,060 And this type of subunit does not bind 66 00:04:54,060 --> 00:04:57,090 nucleotide when in this unloadable conformation. 67 00:05:00,760 --> 00:05:03,495 And so we can draw this. 68 00:05:03,495 --> 00:05:05,120 Here, again, we have the large subunit. 69 00:05:08,430 --> 00:05:11,040 And there's a change in conformation. 70 00:05:11,040 --> 00:05:13,740 And here's the small subunit here. 71 00:05:16,320 --> 00:05:20,970 So what's been found from looking at many crystal 72 00:05:20,970 --> 00:05:26,580 structures is that within the ClpX hexamer, 73 00:05:26,580 --> 00:05:29,820 there's an arrangement of these loadable and unloadable 74 00:05:29,820 --> 00:05:31,140 subunits. 75 00:05:31,140 --> 00:05:40,960 So in many crystals, what's found 76 00:05:40,960 --> 00:05:44,018 is that there's four loadable-- 77 00:05:44,018 --> 00:05:45,310 I'm just going to do with "L"-- 78 00:05:45,310 --> 00:05:55,240 plus two unloadable subunits arranged with about 79 00:05:55,240 --> 00:05:57,040 two-fold symmetry, so LULLUL. 80 00:06:01,960 --> 00:06:04,380 So there's some asymmetry in the subunits. 81 00:06:12,640 --> 00:06:15,130 And so also from these crystal structures, 82 00:06:15,130 --> 00:06:16,840 there's some more observations that we 83 00:06:16,840 --> 00:06:20,380 don't see with just these cartoons of the 6-mer. 84 00:06:20,380 --> 00:06:24,190 So we can learn about how subunits interact, of course, 85 00:06:24,190 --> 00:06:25,975 and this is what's shown. 86 00:06:42,230 --> 00:06:44,480 So if we look at these structures 87 00:06:44,480 --> 00:06:48,200 and consider how these subunits interact, what we find 88 00:06:48,200 --> 00:06:52,060 is that the small subunit of one ClpX-- 89 00:06:52,060 --> 00:06:56,630 or sorry, the small domain of one ClpX subunit interacts 90 00:06:56,630 --> 00:07:01,470 with the large domain of the adjacent ClpX subunit. 91 00:07:01,470 --> 00:07:02,645 And so we can draw this. 92 00:07:06,050 --> 00:07:12,030 Basically, if we consider a large domain-- 93 00:07:12,030 --> 00:07:13,880 and let's say this is subunit 2-- 94 00:07:17,000 --> 00:07:20,160 then what we find is that there's 95 00:07:20,160 --> 00:07:27,890 the small domain and then the large domain 96 00:07:27,890 --> 00:07:29,540 of subunit next door. 97 00:07:29,540 --> 00:07:31,540 So let's call this subunit 1. 98 00:07:34,340 --> 00:07:40,010 So here's our hinge of subunit 1, 99 00:07:40,010 --> 00:07:44,150 and ATP binding happens in here. 100 00:07:44,150 --> 00:07:46,700 So we can think about this arrangement. 101 00:07:46,700 --> 00:07:53,210 And then what's been defined is something called a rigid body. 102 00:07:53,210 --> 00:07:56,690 And so this rigid body is comprised 103 00:07:56,690 --> 00:08:00,590 of the large domain of one subunit 104 00:08:00,590 --> 00:08:03,800 and the small domain of the next. 105 00:08:07,400 --> 00:08:08,240 Rigid body. 106 00:08:23,620 --> 00:08:40,663 So large domain of one ClpX and small domain of another subunit 107 00:08:40,663 --> 00:08:41,330 that's adjacent. 108 00:08:49,710 --> 00:08:55,430 So in thinking about this, we can consider the ClpX hexamer 109 00:08:55,430 --> 00:08:56,960 in another way. 110 00:08:56,960 --> 00:08:59,270 So how I've initially presented it to you 111 00:08:59,270 --> 00:09:04,250 when we introduced these oligomers is just as a 6-mer, 112 00:09:04,250 --> 00:09:04,750 right? 113 00:09:04,750 --> 00:09:06,110 6 subunits. 114 00:09:06,110 --> 00:09:08,750 But another way to think about ClpX 115 00:09:08,750 --> 00:09:14,630 is that it's actually six rigid bodies that 116 00:09:14,630 --> 00:09:18,560 are connected by hinges, where each rigid body has 117 00:09:18,560 --> 00:09:22,760 a component from two subunits, a large domain from one 118 00:09:22,760 --> 00:09:23,840 and a small from another. 119 00:09:26,720 --> 00:09:32,000 And so the hinges are within a single subunit 120 00:09:32,000 --> 00:09:35,690 based on this cartoon where ATP binds. 121 00:09:35,690 --> 00:09:40,310 And so the thinking is that ATP binding and hydrolysis 122 00:09:40,310 --> 00:10:09,190 results in changes in the hinge geometry 123 00:10:09,190 --> 00:10:10,950 and that this change in confirmation 124 00:10:10,950 --> 00:10:14,310 in the hinge with ATP binding and hydrolysis 125 00:10:14,310 --> 00:10:17,580 allows for conformational change in another subunit here. 126 00:10:23,390 --> 00:10:26,000 So six rigid bodies connected by six 127 00:10:26,000 --> 00:10:28,910 hinges, effectively, as opposed to just six 128 00:10:28,910 --> 00:10:30,650 standalone subunits. 129 00:10:30,650 --> 00:10:36,140 Each subunit's communicating with one another here. 130 00:10:36,140 --> 00:10:38,380 So this is pretty complicated, right? 131 00:10:38,380 --> 00:10:42,500 It's another level of sophistication 132 00:10:42,500 --> 00:10:45,530 within this hexamer here. 133 00:10:45,530 --> 00:10:51,380 So what about these loadable and unloadable conformations? 134 00:10:51,380 --> 00:10:53,000 I've told you that in these crystal 135 00:10:53,000 --> 00:10:57,680 structures, what's seen often are these four 136 00:10:57,680 --> 00:11:00,470 loadable and two unloadable subunits 137 00:11:00,470 --> 00:11:03,240 with a particular arrangement. 138 00:11:03,240 --> 00:11:06,890 So we can ask the question, do these individual subunits 139 00:11:06,890 --> 00:11:12,830 maintain the same conformation during these attempts 140 00:11:12,830 --> 00:11:17,900 to denature and translocate polypeptides? 141 00:11:17,900 --> 00:11:20,390 So is one subunit just committed to being 142 00:11:20,390 --> 00:11:25,760 loadable and another subunit committed to being unloadable? 143 00:11:25,760 --> 00:11:27,800 Or did they switch dynamically? 144 00:11:27,800 --> 00:11:29,570 And so recently, there were a number 145 00:11:29,570 --> 00:11:32,390 of studies looking at that. 146 00:11:32,390 --> 00:11:36,960 And effectively, as of a few years ago, 147 00:11:36,960 --> 00:11:38,690 many studies suggest-- 148 00:11:45,371 --> 00:11:59,470 or support switching by a given subunit. 149 00:12:05,240 --> 00:12:09,770 And they also indicate that every ClpX subunit 150 00:12:09,770 --> 00:12:14,330 must bind to ATP at some point during these cycles 151 00:12:14,330 --> 00:12:16,490 for unfolding and translocation. 152 00:12:25,460 --> 00:12:28,490 But they're not all doing it at the same time. 153 00:13:00,350 --> 00:13:02,970 So the way to think about this is 154 00:13:02,970 --> 00:13:05,850 that there's some dynamic interconversion 155 00:13:05,850 --> 00:13:10,530 between these loadable and unloadable subunits 156 00:13:10,530 --> 00:13:13,420 within the hexamer, and somehow-- 157 00:13:13,420 --> 00:13:14,213 yep? 158 00:13:14,213 --> 00:13:15,630 AUDIENCE: I just want to ask you-- 159 00:13:15,630 --> 00:13:16,380 ELIZABETH NOLAN: Going to make trouble? 160 00:13:16,380 --> 00:13:17,210 JOANNE STUBBE: --a question. 161 00:13:17,210 --> 00:13:18,030 Yeah. 162 00:13:18,030 --> 00:13:20,550 So when you have all these structures, 163 00:13:20,550 --> 00:13:22,858 are they all with an ATP analogue? 164 00:13:22,858 --> 00:13:24,900 ELIZABETH NOLAN: I don't know the answer to that. 165 00:13:24,900 --> 00:13:25,650 JOANNE STUBBE: OK. 166 00:13:25,650 --> 00:13:30,000 Because ATP analogues have wide-- 167 00:13:30,000 --> 00:13:35,730 you guys have already seen ADPNP or ADPCH2P They really 168 00:13:35,730 --> 00:13:38,940 have very different properties when you study these, the ATPs. 169 00:13:38,940 --> 00:13:42,290 So if these-- and probably they don't have ATP because they 170 00:13:42,290 --> 00:13:42,790 probably-- 171 00:13:42,790 --> 00:13:43,140 ELIZABETH NOLAN: Right. 172 00:13:43,140 --> 00:13:44,100 They want to get a stable-- 173 00:13:44,100 --> 00:13:45,690 JOANNE STUBBE: So anyhow, that's something to keep 174 00:13:45,690 --> 00:13:46,470 in the back of your mind. 175 00:13:46,470 --> 00:13:48,637 ELIZABETH NOLAN: So just is this an artifact is what 176 00:13:48,637 --> 00:13:51,750 JoAnne's suggesting from use of a non-hydrolysable ATP 177 00:13:51,750 --> 00:13:52,490 analogue. 178 00:13:52,490 --> 00:13:53,820 JOANNE STUBBE: And there's many examples 179 00:13:53,820 --> 00:13:54,970 of that in the literature. 180 00:13:54,970 --> 00:13:55,720 Everybody uses it. 181 00:13:55,720 --> 00:13:58,303 It's just something you need to keep in the back of your mind. 182 00:13:58,303 --> 00:13:59,460 That's the best we can do. 183 00:13:59,460 --> 00:14:01,043 ELIZABETH NOLAN: So next week, someone 184 00:14:01,043 --> 00:14:06,260 should ask during recitation there, for that. 185 00:14:06,260 --> 00:14:12,810 So what about the mechanical work? 186 00:14:12,810 --> 00:14:18,300 How this is often depicted, in terms of grabbing and pulling 187 00:14:18,300 --> 00:14:22,245 on a polypeptide substrate, is via these rigid bodies. 188 00:14:26,736 --> 00:14:32,010 And we're not going to go into details about this, 189 00:14:32,010 --> 00:14:37,230 but just to describe the typical cartoon picture effectively, 190 00:14:37,230 --> 00:14:44,310 imagine we have some polypeptide that needs to enter 191 00:14:44,310 --> 00:14:45,465 the degradation chamber. 192 00:14:50,950 --> 00:14:55,300 So those pore loops we heard about that 193 00:14:55,300 --> 00:15:09,740 are involved in substrate binding 194 00:15:09,740 --> 00:15:13,670 are in the large domain of ClpX. 195 00:15:13,670 --> 00:15:15,970 So here we have one large domain, 196 00:15:15,970 --> 00:15:21,220 and then we can have the small domain of the adjacent subunit 197 00:15:21,220 --> 00:15:23,360 here. 198 00:15:23,360 --> 00:15:28,550 And just imagine here we have another large domain 199 00:15:28,550 --> 00:15:30,710 with its pore loop. 200 00:15:30,710 --> 00:15:37,370 And then we'd have the adjacent subunit here. 201 00:15:37,370 --> 00:15:41,900 So effectively, it's thought that these pore loops 202 00:15:41,900 --> 00:15:46,040 in the large domains grip the substrate and help 203 00:15:46,040 --> 00:15:49,280 drag the substrate to allow for translocation 204 00:15:49,280 --> 00:15:51,230 into the degradation chamber. 205 00:15:51,230 --> 00:15:54,020 So this would be going to the chamber, that direction 206 00:15:54,020 --> 00:15:55,760 here for that. 207 00:15:55,760 --> 00:15:58,460 So somehow the ATP binding and hydrolysis 208 00:15:58,460 --> 00:16:00,410 is allowing this to occur-- 209 00:16:00,410 --> 00:16:08,060 so to ClpP here for that. 210 00:16:08,060 --> 00:16:10,940 So next week in recitation, you're 211 00:16:10,940 --> 00:16:15,890 going to have a real treat because an expert, Reuben, will 212 00:16:15,890 --> 00:16:18,800 be discussing some single molecule methods that 213 00:16:18,800 --> 00:16:23,150 have been applied to studying this degradation chamber. 214 00:16:23,150 --> 00:16:25,970 So bring your questions to him because he really 215 00:16:25,970 --> 00:16:31,100 knows what is state of the field right now for this. 216 00:16:31,100 --> 00:16:36,310 So we've talked a lot about how the substrate needs to get in. 217 00:16:36,310 --> 00:16:37,670 We have the SSRI tag. 218 00:16:37,670 --> 00:16:40,520 We have all of this ATP consumption unfolding, 219 00:16:40,520 --> 00:16:42,200 translocation by ClpX. 220 00:16:42,200 --> 00:16:45,770 And then we talked about the serine protease mechanism 221 00:16:45,770 --> 00:16:49,130 in terms of how peptides are degraded in the chamber. 222 00:16:49,130 --> 00:16:53,420 So then the final question just going to touch upon is, 223 00:16:53,420 --> 00:16:56,810 how does the polypeptide that's been degraded 224 00:16:56,810 --> 00:16:59,630 get out of the chamber? 225 00:16:59,630 --> 00:17:09,410 So ClpXP will give products that are 226 00:17:09,410 --> 00:17:16,010 7 to 8 amino acids in length, so short polypeptides. 227 00:17:21,790 --> 00:17:23,155 So how are they released? 228 00:17:27,040 --> 00:17:29,530 And we can think about two possibilities 229 00:17:29,530 --> 00:17:32,420 for how these polypeptides are released. 230 00:17:32,420 --> 00:17:38,500 One is that they're released through the axial pores. 231 00:17:38,500 --> 00:17:42,340 So somehow those pores that allow polypeptide substrate 232 00:17:42,340 --> 00:17:47,140 to go in also allow product fragments to go out. 233 00:17:47,140 --> 00:17:52,450 And then the second option is that there's release 234 00:17:52,450 --> 00:18:06,490 through transient side pores between the ClpP 7-mers. 235 00:18:09,120 --> 00:18:19,080 So effectively, if we imagine coming back to our ClpP, 236 00:18:19,080 --> 00:18:21,120 we have a 7-mer-- 237 00:18:21,120 --> 00:18:30,930 back-to-back 7-mers, do the fragments 238 00:18:30,930 --> 00:18:36,090 come out, say, of the hole? 239 00:18:36,090 --> 00:18:39,270 Or somehow do they come out from this region here? 240 00:18:44,330 --> 00:18:47,460 To the best of my knowledge, this is a bit unclear, 241 00:18:47,460 --> 00:18:51,210 and I don't think they're mutually exclusive. 242 00:18:51,210 --> 00:18:52,820 So questions have come up. 243 00:18:52,820 --> 00:18:55,700 If they're to come out of an axial pore, 244 00:18:55,700 --> 00:18:58,190 does that mean ClpX has to be dissociated? 245 00:19:01,100 --> 00:19:03,500 In terms of this equator region, there 246 00:19:03,500 --> 00:19:06,380 are structures showing that this degradation 247 00:19:06,380 --> 00:19:07,670 chamber can breathe. 248 00:19:07,670 --> 00:19:11,900 And there's a picture of that in the posted notes from Friday 249 00:19:11,900 --> 00:19:14,810 where you can see opening here. 250 00:19:14,810 --> 00:19:16,580 And there has been some experiments 251 00:19:16,580 --> 00:19:22,520 done where people have put cysteines in this region 252 00:19:22,520 --> 00:19:24,150 by site-directed mutagenesis. 253 00:19:24,150 --> 00:19:26,850 So you can imagine, for instance, 254 00:19:26,850 --> 00:19:32,210 if you have a cysteine here and a cysteine here, 255 00:19:32,210 --> 00:19:38,180 and you oxidize to form a disulfide such that those two 256 00:19:38,180 --> 00:19:40,040 7-mers are locked together. 257 00:19:40,040 --> 00:19:44,210 You can ask, if we load the chamber with small polypeptides 258 00:19:44,210 --> 00:19:48,860 and we have these effectively cross-linked by disulfides, 259 00:19:48,860 --> 00:19:50,900 can the polypeptides get out? 260 00:19:50,900 --> 00:19:55,270 And then if we reduce this to have them no longer attached 261 00:19:55,270 --> 00:19:58,660 to one another, do those polypeptides 262 00:19:58,660 --> 00:20:01,280 stay put or not there? 263 00:20:01,280 --> 00:20:03,800 Those experiments gave some evidence 264 00:20:03,800 --> 00:20:08,510 for release of peptides through this region here, 265 00:20:08,510 --> 00:20:10,760 but there's also evidence for release of peptides 266 00:20:10,760 --> 00:20:12,680 through the pore. 267 00:20:12,680 --> 00:20:14,992 And in terms of cartoon depictions. 268 00:20:14,992 --> 00:20:16,950 In the lecture notes, if you take a close look, 269 00:20:16,950 --> 00:20:19,620 you'll see that both come out there. 270 00:20:19,620 --> 00:20:23,360 So I'd say if you're curious about that, 271 00:20:23,360 --> 00:20:25,430 you can read some of the literature 272 00:20:25,430 --> 00:20:29,960 and come to some own conclusions. 273 00:20:29,960 --> 00:20:38,600 One last point on the Clp system before we move on to module 4, 274 00:20:38,600 --> 00:20:41,600 you should just be aware that there's other Clp family 275 00:20:41,600 --> 00:20:43,340 members. 276 00:20:43,340 --> 00:20:50,990 So not only ClpX and P. And so in the Clp system-- 277 00:20:50,990 --> 00:20:53,240 actually, I'm going to make one other point after this 278 00:20:53,240 --> 00:20:56,165 too, about degradation chambers. 279 00:21:01,710 --> 00:21:07,950 So there are players ClpA, ClpB, in addition to ClpX. 280 00:21:10,860 --> 00:21:20,945 So these are all three different AAA+ ATPases. 281 00:21:24,600 --> 00:21:28,770 And you've actually encountered ClpB last week. 282 00:21:28,770 --> 00:21:33,450 So this is HSP 100, which came up in question 2 on the exam 283 00:21:33,450 --> 00:21:36,210 there by another name. 284 00:21:36,210 --> 00:21:39,380 And then in addition to ClpP, there's 285 00:21:39,380 --> 00:21:46,230 also ClpS and some other players here for that. 286 00:21:46,230 --> 00:21:52,200 They each have their own personality within protein 287 00:21:52,200 --> 00:21:55,430 quality control here for that. 288 00:22:02,540 --> 00:22:06,890 And then we've only looked at this degradation chamber 289 00:22:06,890 --> 00:22:09,642 from bacteria. 290 00:22:09,642 --> 00:22:11,100 You might want to ask the question, 291 00:22:11,100 --> 00:22:16,380 what happens in other organisms? 292 00:22:16,380 --> 00:22:20,420 And the answer is that the complexity varies and systems 293 00:22:20,420 --> 00:22:25,280 become tremendously more complex as you move from bacteria 294 00:22:25,280 --> 00:22:27,360 into eukaryotes there. 295 00:22:27,360 --> 00:22:32,670 And so if we consider the different degradation chambers, 296 00:22:32,670 --> 00:22:34,370 what do we see? 297 00:22:34,370 --> 00:22:38,930 So we find these proteasomes in all forms of life. 298 00:22:38,930 --> 00:22:42,020 And as I just said, the level of complexity 299 00:22:42,020 --> 00:22:44,820 varies depending on the organism. 300 00:22:44,820 --> 00:22:51,410 And so what we've seen with ClpP is the most simple system 301 00:22:51,410 --> 00:22:58,380 where we have two rings that have only one type of subunit. 302 00:22:58,380 --> 00:23:00,570 So just say E. coli. 303 00:23:00,570 --> 00:23:01,730 One type of subunit. 304 00:23:17,300 --> 00:23:20,000 What happens if we go to archae? 305 00:23:20,000 --> 00:23:23,570 We find that we have four rings, each of which is 7-mer. 306 00:23:34,770 --> 00:23:39,160 And these four rings include two different types of subunits. 307 00:23:39,160 --> 00:23:41,430 So I'll call these alpha and beta. 308 00:23:41,430 --> 00:23:46,740 So what we find is that there's a 7-mer of 7 alpha subunits, 309 00:23:46,740 --> 00:23:50,610 then 7-mers that have 7 beta subunits, and here 310 00:23:50,610 --> 00:23:51,555 a 7-mer with alpha. 311 00:23:55,800 --> 00:24:04,465 So we see two types of subunit and four rings. 312 00:24:11,080 --> 00:24:14,080 So then what about yeast? 313 00:24:14,080 --> 00:24:15,460 Tremendously complex. 314 00:24:15,460 --> 00:24:20,440 So we have this architecture again 315 00:24:20,440 --> 00:24:28,030 of four rings, an organized alpha, beta, beta, alpha. 316 00:24:33,720 --> 00:24:42,300 But what we find in this case is that in each of these-- 317 00:24:42,300 --> 00:24:44,580 I'm not going to draw it like that, but each of these 318 00:24:44,580 --> 00:24:45,830 have seven different subunits. 319 00:24:55,250 --> 00:24:56,960 There's a depiction of this in the notes. 320 00:25:08,770 --> 00:25:14,550 So just imagine-- how does this get assembled? 321 00:25:14,550 --> 00:25:15,810 I have no clue. 322 00:25:15,810 --> 00:25:18,300 But somehow each of these heptamers 323 00:25:18,300 --> 00:25:21,000 has to be assembled with seven different subunits. 324 00:25:21,000 --> 00:25:23,910 And then they're put together in this series of four rings. 325 00:25:23,910 --> 00:25:26,370 And then as you'll see after spring break in JoAnne's 326 00:25:26,370 --> 00:25:29,340 section, the eukaryotic proteasome 327 00:25:29,340 --> 00:25:33,600 has this 19S regulatory particle that's 328 00:25:33,600 --> 00:25:36,840 involved in recognizing condemned proteins that 329 00:25:36,840 --> 00:25:38,760 have polyubiquitin chains. 330 00:25:38,760 --> 00:25:44,020 And compared to the ClpX ATPase, it's much, much more complex. 331 00:25:44,020 --> 00:25:45,630 So there's many different proteins 332 00:25:45,630 --> 00:25:50,220 that constitute this necessary part of the machine. 333 00:25:50,220 --> 00:25:54,450 But there is a hexamer, ATPase hexamer, within there 334 00:25:54,450 --> 00:25:57,660 to facilitate translocation of the polypeptide 335 00:25:57,660 --> 00:26:01,260 into the degradation chamber. 336 00:26:01,260 --> 00:26:05,222 So some of this will come back again 337 00:26:05,222 --> 00:26:06,555 in the latter half of the class. 338 00:26:09,170 --> 00:26:14,980 So with that, we're going to close on degradation and move 339 00:26:14,980 --> 00:26:21,400 into module 4, which is focused on macromolecular machines that 340 00:26:21,400 --> 00:26:25,120 are involved in the biosynthesis of natural products, 341 00:26:25,120 --> 00:26:29,360 specifically polyketides and nonribosomal peptides. 342 00:26:29,360 --> 00:26:32,920 And so we're completely taking a loop back 343 00:26:32,920 --> 00:26:35,920 to thinking about a biological polymerization, 344 00:26:35,920 --> 00:26:38,470 like what we were thinking with the ribosome 345 00:26:38,470 --> 00:26:42,700 from the process of breaking down a polypeptide. 346 00:26:42,700 --> 00:26:45,760 And so where are we going? 347 00:26:49,510 --> 00:26:51,220 We can think about assembly lines, 348 00:26:51,220 --> 00:26:54,160 although this is a helpful way on the board 349 00:26:54,160 --> 00:26:55,413 to think about these systems. 350 00:26:55,413 --> 00:26:57,080 But it's not really what they look like. 351 00:26:57,080 --> 00:27:00,520 And you'll learn about that in recitation this week. 352 00:27:00,520 --> 00:27:01,020 Yeah? 353 00:27:01,020 --> 00:27:02,844 AUDIENCE: Could you explain the interaction 354 00:27:02,844 --> 00:27:06,057 between ATP and the hinge area? 355 00:27:06,057 --> 00:27:06,890 ELIZABETH NOLAN: OK. 356 00:27:06,890 --> 00:27:11,510 So the ATP binding site is just rewinding here 357 00:27:11,510 --> 00:27:13,130 in that hinge region. 358 00:27:13,130 --> 00:27:15,500 And there's going to be conformational change 359 00:27:15,500 --> 00:27:19,100 in the hinge with ATP binding and hydrolysis there. 360 00:27:19,100 --> 00:27:23,420 And that's sufficient in terms of the level of detail 361 00:27:23,420 --> 00:27:23,960 for this. 362 00:27:23,960 --> 00:27:28,340 But the main thing to keep in mind, each subunit binds ATP. 363 00:27:28,340 --> 00:27:31,310 But on the basis of the information gathered 364 00:27:31,310 --> 00:27:33,620 with the caveats JoAnne brought up, 365 00:27:33,620 --> 00:27:38,600 different subunits bind ATP at different times in the cycle. 366 00:27:38,600 --> 00:27:39,512 AUDIENCE: OK. 367 00:27:39,512 --> 00:27:41,180 Thank you. 368 00:27:41,180 --> 00:27:43,490 ELIZABETH NOLAN: And changes in this subunit, 369 00:27:43,490 --> 00:27:45,530 conformational changes that result from that, 370 00:27:45,530 --> 00:27:49,945 can be translated to the next door subunit here. 371 00:27:49,945 --> 00:27:51,310 AUDIENCE: OK. 372 00:27:51,310 --> 00:27:53,190 ELIZABETH NOLAN: OK. 373 00:27:53,190 --> 00:27:56,340 So where are we going? 374 00:27:56,340 --> 00:28:02,100 By a week from now, you should have a good handle 375 00:28:02,100 --> 00:28:04,980 on how to think about the biosynthesis of structures 376 00:28:04,980 --> 00:28:08,310 like erythromycin, of penicillin. 377 00:28:08,310 --> 00:28:12,450 These are products of assembly lines. 378 00:28:12,450 --> 00:28:16,950 And so where we'll go is with a brief overview of fatty acid 379 00:28:16,950 --> 00:28:20,880 biosynthesis and then look into polyketide synthase 380 00:28:20,880 --> 00:28:25,510 and nonribosomal peptide synthetase assembly lines here. 381 00:28:25,510 --> 00:28:26,970 And then some case studies. 382 00:28:26,970 --> 00:28:32,850 So on the topic of ATP, where we just went back to with ClpX, 383 00:28:32,850 --> 00:28:36,090 just taking a look here, what do you 384 00:28:36,090 --> 00:28:40,450 know about these systems in ATP by the names? 385 00:28:40,450 --> 00:28:49,400 This is just a little language use and definition. 386 00:28:49,400 --> 00:28:52,832 So there's a subtle difference here. 387 00:28:52,832 --> 00:28:53,998 What's the difference? 388 00:28:53,998 --> 00:28:55,540 AUDIENCE: Synthase versus synthetase? 389 00:28:55,540 --> 00:28:56,300 ELIZABETH NOLAN: Yeah. 390 00:28:56,300 --> 00:28:58,217 And what does that tell you right off the bat? 391 00:29:01,112 --> 00:29:02,086 About ATP. 392 00:29:14,750 --> 00:29:18,380 So it's a subtlety, right? 393 00:29:18,380 --> 00:29:20,510 Synthase is a general term. 394 00:29:20,510 --> 00:29:23,380 Synthetase indicates ATP is involved. 395 00:29:23,380 --> 00:29:25,485 So as we'll see, these nonribosomal peptide 396 00:29:25,485 --> 00:29:27,662 synthetases employ ATP. 397 00:29:27,662 --> 00:29:29,870 And we're going to see chemistry very similar to what 398 00:29:29,870 --> 00:29:32,840 you saw with the aminoacyl-tRNA synthetases 399 00:29:32,840 --> 00:29:35,720 in terms of activating amino acid monomers. 400 00:29:35,720 --> 00:29:40,640 But in this case, the machine is forming a nonribosomal peptide 401 00:29:40,640 --> 00:29:45,320 rather than a ribosomal peptide here. 402 00:29:45,320 --> 00:29:49,790 If you are not familiar with fatty acid biosynthesis, 403 00:29:49,790 --> 00:29:52,550 I highly encourage you to go do some review, 404 00:29:52,550 --> 00:29:55,880 either from your 5.07 notes last term if you were in the class 405 00:29:55,880 --> 00:29:57,740 or from a biochemistry book. 406 00:29:57,740 --> 00:30:00,860 And there'll be some additional slides of overview information 407 00:30:00,860 --> 00:30:01,820 posted online. 408 00:30:01,820 --> 00:30:04,400 So we'll just touch upon it today but not 409 00:30:04,400 --> 00:30:08,090 go into tremendous detail here. 410 00:30:08,090 --> 00:30:12,155 So what are our questions for this module? 411 00:30:12,155 --> 00:30:14,480 I think for most everyone in the room, 412 00:30:14,480 --> 00:30:18,050 this module will contain the most new information 413 00:30:18,050 --> 00:30:19,550 from the standpoint of a new system 414 00:30:19,550 --> 00:30:22,740 compared to what we've talked about so far. 415 00:30:22,740 --> 00:30:27,500 So what are polyketides and how are these molecules 416 00:30:27,500 --> 00:30:31,430 biosynthesized by polyketide synthases? 417 00:30:31,430 --> 00:30:34,130 What are nonribosomal peptides and how are they 418 00:30:34,130 --> 00:30:37,640 made by these machines called nonribosomal peptide 419 00:30:37,640 --> 00:30:38,930 synthetases? 420 00:30:38,930 --> 00:30:42,230 And what we're going to look at is the assembly line 421 00:30:42,230 --> 00:30:47,420 organization, so effectively the organization of domains that 422 00:30:47,420 --> 00:30:52,620 provide these linear polymers. 423 00:30:52,620 --> 00:30:57,050 So what is the assembly line organization and logic for PKS? 424 00:30:57,050 --> 00:30:59,720 And likewise for NRPS. 425 00:30:59,720 --> 00:31:02,540 And then we can ask, how can a given assembly 426 00:31:02,540 --> 00:31:07,250 line for a given PKS or NRPS natural product 427 00:31:07,250 --> 00:31:11,390 be basically predicted from the structure 428 00:31:11,390 --> 00:31:12,530 of the natural product? 429 00:31:12,530 --> 00:31:14,900 So you should be able to work back and forth in terms 430 00:31:14,900 --> 00:31:16,550 of looking at a structure and coming up 431 00:31:16,550 --> 00:31:19,670 with a biosynthetic prediction and also seeing 432 00:31:19,670 --> 00:31:22,190 biosynthetic machinery and getting a sense as to what 433 00:31:22,190 --> 00:31:27,290 that small molecule metabolite's backbone might look like. 434 00:31:27,290 --> 00:31:30,290 How are these studied experimentally? 435 00:31:30,290 --> 00:31:32,660 And we'll look at the biosynthesis 436 00:31:32,660 --> 00:31:35,870 of a molecule called enterobactin as a case study. 437 00:31:35,870 --> 00:31:38,240 And so one thing I'll just point out right now 438 00:31:38,240 --> 00:31:42,380 is that these synthases and synthetases do not 439 00:31:42,380 --> 00:31:43,880 look like an assembly line. 440 00:31:43,880 --> 00:31:46,910 And we'll draw domains in a linear order which really 441 00:31:46,910 --> 00:31:50,000 facilitates thinking about the chemistry, 442 00:31:50,000 --> 00:31:54,710 but the structures are not just a line of domains or proteins 443 00:31:54,710 --> 00:31:56,430 next door to one another. 444 00:31:56,430 --> 00:31:58,370 And this week in recitation, you'll 445 00:31:58,370 --> 00:32:04,370 get to see some cryo-EM studies on fatty acid synthase and 446 00:32:04,370 --> 00:32:06,650 related machines there which will give you 447 00:32:06,650 --> 00:32:11,310 a sense of their dynamics. 448 00:32:11,310 --> 00:32:14,120 So just a review. 449 00:32:14,120 --> 00:32:18,260 If we think about template-dependent 450 00:32:18,260 --> 00:32:20,720 polymerizations in biology, we're 451 00:32:20,720 --> 00:32:24,890 all familiar with DNA replication, transcription, 452 00:32:24,890 --> 00:32:27,050 and translation. 453 00:32:27,050 --> 00:32:29,690 And what you'll see in this unit is 454 00:32:29,690 --> 00:32:32,600 that these template-driven polymerizations 455 00:32:32,600 --> 00:32:37,730 occur in the biosynthesis of natural products here. 456 00:32:37,730 --> 00:32:39,740 And effectively, these assembly lines, in a way, 457 00:32:39,740 --> 00:32:42,080 provide this template. 458 00:32:42,080 --> 00:32:45,260 So they're small molecules being biosynthesized 459 00:32:45,260 --> 00:32:51,840 by microbes using some pretty amazing machinery. 460 00:32:51,840 --> 00:32:53,780 So when we think about template-driven 461 00:32:53,780 --> 00:32:57,950 polymerizations, we think about an initiation process, 462 00:32:57,950 --> 00:33:00,560 elongation process, and termination. 463 00:33:00,560 --> 00:33:03,680 We saw that with a translation cycle. 464 00:33:03,680 --> 00:33:10,160 And we'll see the same type of systems here. 465 00:33:10,160 --> 00:33:14,030 So what does some of these structures look like? 466 00:33:14,030 --> 00:33:16,490 Here are just some examples on the top 467 00:33:16,490 --> 00:33:21,440 of polyketides, two examples. 468 00:33:21,440 --> 00:33:24,950 They look very different at first glance, and they are. 469 00:33:24,950 --> 00:33:27,350 So we have tetracycline. 470 00:33:27,350 --> 00:33:30,830 We have four fused 6-membered rings. 471 00:33:30,830 --> 00:33:34,610 It's an aromatic polyketide, an antibiotic. 472 00:33:34,610 --> 00:33:39,410 We have this erythromycin here, which is a macrolide. 473 00:33:39,410 --> 00:33:42,350 We encountered macrolines in the translation section 474 00:33:42,350 --> 00:33:46,850 because they bind the ribosome, another type of antibiotic. 475 00:33:46,850 --> 00:33:51,890 If we look at some nonribosomal peptides, all of that 476 00:33:51,890 --> 00:33:53,330 can be used clinically. 477 00:33:53,330 --> 00:33:54,980 We see the penicillins. 478 00:33:54,980 --> 00:34:01,940 So we have a 4 or 5 fused ring system here, a beta-lactam. 479 00:34:01,940 --> 00:34:06,320 This comes from three amino acid building blocks initially. 480 00:34:06,320 --> 00:34:08,060 We have vancomycin. 481 00:34:08,060 --> 00:34:10,730 This is an antibiotic of last resort. 482 00:34:10,730 --> 00:34:14,350 And this structure looks really quite complicated, 483 00:34:14,350 --> 00:34:19,540 but what we'll see is that it's based on seven 484 00:34:19,540 --> 00:34:21,050 proteogenic amino acids. 485 00:34:21,050 --> 00:34:23,650 So it's the 7-mer peptide backbone 486 00:34:23,650 --> 00:34:29,530 that gives rise to this structure here for that. 487 00:34:29,530 --> 00:34:31,270 And then we see there's some sugars, 488 00:34:31,270 --> 00:34:35,920 so these can be put on by other enzymes here. 489 00:34:35,920 --> 00:34:40,090 So on top we see a lot of ketones and OH groups. 490 00:34:40,090 --> 00:34:44,710 Those are good hints that maybe polyketide logic is being used. 491 00:34:44,710 --> 00:34:47,510 Here we see a number of peptide bonds, 492 00:34:47,510 --> 00:34:53,550 amide bonds, a good indicator of NRPS at play. 493 00:34:53,550 --> 00:34:58,860 And here, just to point out, these systems get very complex. 494 00:34:58,860 --> 00:35:02,250 And there's natural products out there 495 00:35:02,250 --> 00:35:05,430 that are biosynthesized from a combination 496 00:35:05,430 --> 00:35:10,260 of polyketide synthase logic and nonribosomal peptide synthetase 497 00:35:10,260 --> 00:35:11,820 logic here. 498 00:35:11,820 --> 00:35:15,960 These include molecules like yersiniabactin. 499 00:35:15,960 --> 00:35:19,080 This is an iron chelator produced by Yersinia pestis, 500 00:35:19,080 --> 00:35:22,640 and some pathogenic E. coli, this immunosuppressant 501 00:35:22,640 --> 00:35:26,070 rapamycin as examples. 502 00:35:26,070 --> 00:35:28,860 So as we move forward, I put a lot 503 00:35:28,860 --> 00:35:32,520 of structures of small molecule metabolites in the slides. 504 00:35:32,520 --> 00:35:34,170 You can go back and use them as a way 505 00:35:34,170 --> 00:35:35,910 to study and try to make predictions 506 00:35:35,910 --> 00:35:39,120 about what is the machinery at play, 507 00:35:39,120 --> 00:35:42,120 for instance, to give all of these heterocycles? 508 00:35:42,120 --> 00:35:43,860 How are those made? 509 00:35:43,860 --> 00:35:47,310 We'll see the assembly line does that. 510 00:35:47,310 --> 00:35:51,060 So what organisms produce these molecules? 511 00:35:51,060 --> 00:35:54,870 Largely, bacteria and fungi. 512 00:35:54,870 --> 00:35:59,550 And there are some correlations out there, I'll just point out, 513 00:35:59,550 --> 00:36:05,480 related to genome size and the number 514 00:36:05,480 --> 00:36:07,970 of metabolites being made. 515 00:36:07,970 --> 00:36:12,380 So bioinformatics guides a lot of current studies 516 00:36:12,380 --> 00:36:15,320 of the biosynthesis of these types of molecules. 517 00:36:15,320 --> 00:36:18,500 So you can imagine that you sequence a genome. 518 00:36:18,500 --> 00:36:22,100 You have some information about gene clusters. 519 00:36:22,100 --> 00:36:23,960 So these are groups of genes where 520 00:36:23,960 --> 00:36:28,100 the proteins work together to biosynthesize the molecule. 521 00:36:28,100 --> 00:36:32,330 And often, the genes that encode proteins in these metabolites 522 00:36:32,330 --> 00:36:34,670 are clustered. 523 00:36:34,670 --> 00:36:38,750 And so bioinformatics approaches can help find these. 524 00:36:38,750 --> 00:36:44,060 What's found is that for bacteria, some phyla 525 00:36:44,060 --> 00:36:48,950 are more prolific producers of these molecules than others. 526 00:36:48,950 --> 00:36:53,090 And what's been shown in a general way 527 00:36:53,090 --> 00:36:56,480 is that organisms with small genomes-- 528 00:36:56,480 --> 00:36:58,700 so something like E. coli-- 529 00:36:58,700 --> 00:37:01,730 produce fewer of these metabolites. 530 00:37:01,730 --> 00:37:03,110 That's not to say none. 531 00:37:03,110 --> 00:37:05,630 So enterobactin, which we'll look at for a case study, 532 00:37:05,630 --> 00:37:07,640 is made by E. coli. 533 00:37:07,640 --> 00:37:09,920 But they don't make as many. 534 00:37:09,920 --> 00:37:17,130 And effectively, organisms with larger genomes produce more. 535 00:37:17,130 --> 00:37:18,860 And so here is just a correlation 536 00:37:18,860 --> 00:37:20,750 between the number of genes. 537 00:37:20,750 --> 00:37:22,640 And the genome size of the organism 538 00:37:22,640 --> 00:37:28,940 where they see around 3 Mb, there's a switch here. 539 00:37:28,940 --> 00:37:30,445 Often, these molecules-- yeah? 540 00:37:30,445 --> 00:37:31,820 AUDIENCE: Is there any hypotheses 541 00:37:31,820 --> 00:37:34,070 about an evolutionary driving factor 542 00:37:34,070 --> 00:37:35,630 for the development of this machinery 543 00:37:35,630 --> 00:37:39,328 and why it correlates to genome size? 544 00:37:39,328 --> 00:37:41,120 ELIZABETH NOLAN: If there is, I don't know. 545 00:37:41,120 --> 00:37:45,050 I don't think about evolution very well, quite frankly. 546 00:37:45,050 --> 00:37:48,980 What is thought is that many of these molecules 547 00:37:48,980 --> 00:37:51,290 are thought to be involved in defense 548 00:37:51,290 --> 00:37:54,290 and that an organism with a smaller genome size 549 00:37:54,290 --> 00:37:55,730 uses other strategies. 550 00:37:55,730 --> 00:37:59,150 And so for instance, E. coli, which 551 00:37:59,150 --> 00:38:01,460 I cited as a small genome, will use 552 00:38:01,460 --> 00:38:05,670 a number of ribosomal peptides as defense molecules 553 00:38:05,670 --> 00:38:08,510 that get post-translationally modified after the fact. 554 00:38:08,510 --> 00:38:12,170 But why that organism chooses to do that versus say something 555 00:38:12,170 --> 00:38:16,430 like Streptomyces that produces many, many different natural 556 00:38:16,430 --> 00:38:19,190 products, I'm not sure about that. 557 00:38:24,170 --> 00:38:29,600 So let's look at an example of a gene cluster, 558 00:38:29,600 --> 00:38:33,590 just so you get a sense of how much machinery 559 00:38:33,590 --> 00:38:39,020 is required to do the full biosynthesis of a molecule. 560 00:38:39,020 --> 00:38:43,190 So this is for a nonribosomal peptide shown here. 561 00:38:43,190 --> 00:38:45,080 It has some structural similarities 562 00:38:45,080 --> 00:38:49,520 to the vancomycin we saw on a prior slide, 563 00:38:49,520 --> 00:38:52,820 and it is a member of the vancomycin family. 564 00:38:52,820 --> 00:38:57,530 So this gene cluster for the biosynthesis of this metabolite 565 00:38:57,530 --> 00:39:02,330 contains 30 different genes and is depicted here. 566 00:39:02,330 --> 00:39:05,210 So each one of these arrows indicates an open reading 567 00:39:05,210 --> 00:39:06,330 frame. 568 00:39:06,330 --> 00:39:10,860 So each one begins with a start, ends with a stop codon. 569 00:39:10,860 --> 00:39:14,300 And it's assumed to be the coding sequence of the gene. 570 00:39:14,300 --> 00:39:17,870 And so what is encoded in these 30 genes? 571 00:39:17,870 --> 00:39:21,290 Well, first there are the genes for what 572 00:39:21,290 --> 00:39:23,270 we call the assembly line. 573 00:39:23,270 --> 00:39:25,610 And if it isn't clear what assembly line means, 574 00:39:25,610 --> 00:39:28,430 as we move forward through this week, it will be. 575 00:39:28,430 --> 00:39:33,650 So there's genes required to make the 7-mer polypeptide 576 00:39:33,650 --> 00:39:35,060 backbone. 577 00:39:35,060 --> 00:39:38,750 There's genes required for modification of the backbone. 578 00:39:38,750 --> 00:39:43,020 So how do these sugars get attached, for instance? 579 00:39:43,020 --> 00:39:45,650 Those are going to be some tailoring enzymes. 580 00:39:45,650 --> 00:39:47,640 And then if you take a close look, 581 00:39:47,640 --> 00:39:51,500 there's a number of non-proteinogenic amino acids 582 00:39:51,500 --> 00:39:53,720 in this molecule, and that means they 583 00:39:53,720 --> 00:39:55,380 have to come from somewhere. 584 00:39:55,380 --> 00:39:57,890 And so this gene cluster also includes 585 00:39:57,890 --> 00:40:02,810 genes that are required for the biosynthesis of those monomers. 586 00:40:02,810 --> 00:40:05,600 So there's a lot of effort going in to making 587 00:40:05,600 --> 00:40:09,140 this molecule by some organism. 588 00:40:09,140 --> 00:40:11,930 And so presumably, under some set of conditions, 589 00:40:11,930 --> 00:40:12,800 it's important. 590 00:40:16,390 --> 00:40:24,130 So moving towards the chemistry, with that background in hand, 591 00:40:24,130 --> 00:40:27,490 what are some points to make? 592 00:40:27,490 --> 00:40:34,660 So what we'll learn and see is that the assembly lines that 593 00:40:34,660 --> 00:40:39,250 produce the polyketides and nonribosomal peptides 594 00:40:39,250 --> 00:40:41,240 are macromolecular machines. 595 00:40:41,240 --> 00:40:44,440 So there's dedicated macromolecular machines 596 00:40:44,440 --> 00:40:49,960 for the biosynthesis of these secondary metabolites. 597 00:40:49,960 --> 00:40:52,510 And so what are secondary metabolites 598 00:40:52,510 --> 00:40:56,120 versus a primary metabolite? 599 00:40:56,120 --> 00:40:57,970 So what's a primary metabolite? 600 00:41:08,530 --> 00:41:11,930 AUDIENCE: I'm not even totally sure how to define metabolites. 601 00:41:11,930 --> 00:41:14,470 Isn't metabolites what goes in? 602 00:41:14,470 --> 00:41:17,180 Or what comes out? 603 00:41:20,269 --> 00:41:21,311 ELIZABETH NOLAN: Rebecca? 604 00:41:21,311 --> 00:41:24,490 AUDIENCE: Or easily produced directly from the materials 605 00:41:24,490 --> 00:41:27,400 the cell's consuming? 606 00:41:27,400 --> 00:41:30,820 ELIZABETH NOLAN: So presumably, the cell 607 00:41:30,820 --> 00:41:33,160 needs to get materials to biosynthesize 608 00:41:33,160 --> 00:41:35,320 the secondary metabolites too, right? 609 00:41:35,320 --> 00:41:37,300 Somewhere, these amino acid monomers 610 00:41:37,300 --> 00:41:40,660 or the monomers that are used for polyketide synthetase 611 00:41:40,660 --> 00:41:41,370 need to-- 612 00:41:41,370 --> 00:41:43,690 they'd have to come from somewhere, right? 613 00:41:43,690 --> 00:41:46,550 So are primary metabolites important for growth? 614 00:41:46,550 --> 00:41:47,225 AUDIENCE: Yes. 615 00:41:47,225 --> 00:41:48,100 ELIZABETH NOLAN: Yes. 616 00:41:48,100 --> 00:41:50,530 Development? 617 00:41:50,530 --> 00:41:51,270 Reproduction? 618 00:41:51,270 --> 00:41:52,170 AUDIENCE: Yes. 619 00:41:52,170 --> 00:41:53,378 ELIZABETH NOLAN: Yeah, right. 620 00:41:53,378 --> 00:41:54,960 Under normal conditions, right? 621 00:41:54,960 --> 00:41:57,540 We're in trouble if we don't have our primary metabolites 622 00:41:57,540 --> 00:42:02,070 there, whether they're ingested or biosynthesized. 623 00:42:02,070 --> 00:42:03,960 What about a secondary metabolite? 624 00:42:03,960 --> 00:42:05,100 Just taking that-- 625 00:42:05,100 --> 00:42:06,195 AUDIENCE: I'm guessing it's not necessary. 626 00:42:06,195 --> 00:42:07,854 AUDIENCE: --something we can make from primary metabolites? 627 00:42:07,854 --> 00:42:09,140 ELIZABETH NOLAN: No. 628 00:42:09,140 --> 00:42:10,650 Well, you can. 629 00:42:10,650 --> 00:42:11,770 You can. 630 00:42:11,770 --> 00:42:13,393 So a secondary-- 631 00:42:13,393 --> 00:42:14,310 AUDIENCE: --necessary? 632 00:42:14,310 --> 00:42:15,227 ELIZABETH NOLAN: Yeah. 633 00:42:15,227 --> 00:42:20,100 A secondary metabolite is not required for normal growth, 634 00:42:20,100 --> 00:42:22,290 development, reproduction. 635 00:42:22,290 --> 00:42:27,690 So for some reason, under some circumstances of need, 636 00:42:27,690 --> 00:42:30,250 these secondary metabolites get produced. 637 00:42:30,250 --> 00:42:33,840 So for some of these antibiotic molecules, maybe 638 00:42:33,840 --> 00:42:36,480 the organism needs to defend itself. 639 00:42:36,480 --> 00:42:39,450 In the case of enterobactin or yersiniabactin, 640 00:42:39,450 --> 00:42:41,880 maybe that organism needs iron. 641 00:42:41,880 --> 00:42:43,770 And so it's producing a molecule that 642 00:42:43,770 --> 00:42:47,880 will help it obtain that there. 643 00:42:47,880 --> 00:42:51,310 So what is going on? 644 00:42:51,310 --> 00:42:54,090 We've seen some pretty complex molecules. 645 00:42:54,090 --> 00:42:59,100 What we're going to see is that these assembly lines convert 646 00:42:59,100 --> 00:43:03,360 simple acid monomers, if it's a polyketide synthase 647 00:43:03,360 --> 00:43:06,700 or amino acid monomers for a nonribosomal peptide 648 00:43:06,700 --> 00:43:09,810 synthetase, into linear polymers. 649 00:43:09,810 --> 00:43:13,350 So we're going to look at template-driven polymerizations 650 00:43:13,350 --> 00:43:16,260 that initially give linear polymers. 651 00:43:16,260 --> 00:43:19,440 And in the case of PKS, this is very similar 652 00:43:19,440 --> 00:43:22,200 to fatty acid biosynthesis. 653 00:43:22,200 --> 00:43:25,530 What we see is that the assembly lines 654 00:43:25,530 --> 00:43:29,100 allow for iterative additions of malonyl and methylmalonyl 655 00:43:29,100 --> 00:43:30,060 units. 656 00:43:30,060 --> 00:43:34,200 And they catalyze carbon-carbon bond formations. 657 00:43:34,200 --> 00:43:38,730 In the case of nonribosomal peptide synthetases, 658 00:43:38,730 --> 00:43:42,990 what we'll see is that these allow for condensations 659 00:43:42,990 --> 00:43:45,810 of amino acids to form peptide bonds 660 00:43:45,810 --> 00:43:49,510 and effectively form nonribosomal polypeptides. 661 00:43:49,510 --> 00:43:53,070 So polypeptide synthesis without the ribosome. 662 00:43:53,070 --> 00:43:57,480 So even though the PKS and NRPS are forming a different type 663 00:43:57,480 --> 00:44:01,770 of bond and that requires different chemistry, what 664 00:44:01,770 --> 00:44:07,050 we'll see is that they use very similar logic. 665 00:44:07,050 --> 00:44:11,730 And just getting the logic sorted out initially 666 00:44:11,730 --> 00:44:13,710 makes life much easier down the road. 667 00:44:13,710 --> 00:44:17,940 So take some time to look over the depictions in the notes 668 00:44:17,940 --> 00:44:21,100 outside of class as we go forward. 669 00:44:21,100 --> 00:44:25,620 So these assembly lines use acyl or aminoacyl thioesters 670 00:44:25,620 --> 00:44:29,040 as the activated monomer units. 671 00:44:29,040 --> 00:44:32,310 So then how do we get from this linear polypeptide 672 00:44:32,310 --> 00:44:34,140 to some more complex structure? 673 00:44:36,750 --> 00:44:40,890 The short message on that is that the, quote, "polymers" 674 00:44:40,890 --> 00:44:42,650 that are produced-- 675 00:44:42,650 --> 00:44:44,040 and they may be short, right? 676 00:44:44,040 --> 00:44:47,790 We just saw-- they are short, 7 amino acids for vancomycin. 677 00:44:47,790 --> 00:44:50,460 They can undergo further elaboration 678 00:44:50,460 --> 00:44:52,620 to give these complex structures. 679 00:44:52,620 --> 00:44:56,490 So there can be tailoring enzymes 680 00:44:56,490 --> 00:45:01,200 that work on the products of the assembly line. 681 00:45:01,200 --> 00:45:03,930 Or there can be domains in the assembly line that 682 00:45:03,930 --> 00:45:08,130 give additional activities that allow for methylation 683 00:45:08,130 --> 00:45:11,940 or cyclization here. 684 00:45:11,940 --> 00:45:19,150 So we can think about fatty acid synthase as a paradigm here. 685 00:45:19,150 --> 00:45:22,560 And so if we think about fatty acid 686 00:45:22,560 --> 00:45:30,330 biosynthesis making some molecule like this oil here, 687 00:45:30,330 --> 00:45:34,830 just as brief overview in the last few minutes of class. 688 00:45:34,830 --> 00:45:38,300 Fatty acids are synthesized by FAS. 689 00:45:38,300 --> 00:45:41,580 And what happens is that there's elongation 690 00:45:41,580 --> 00:45:45,060 by one unit at a time. 691 00:45:45,060 --> 00:45:47,620 And each unit provides two carbons. 692 00:45:47,620 --> 00:45:51,040 So there's two carbon atoms per elongation. 693 00:45:51,040 --> 00:45:53,760 And so hopefully you're all familiar with two ways 694 00:45:53,760 --> 00:45:56,430 to form a carbon-carbon bond, at least related 695 00:45:56,430 --> 00:46:00,840 to biochemistry, one of which is Claisen condensations. 696 00:46:00,840 --> 00:46:04,410 So Claisen condensations allow for carbon-carbon bond 697 00:46:04,410 --> 00:46:09,420 formation and join the units. 698 00:46:09,420 --> 00:46:12,355 To keep in mind, the monomers are always 699 00:46:12,355 --> 00:46:16,350 thioesters, not oxoesters. 700 00:46:16,350 --> 00:46:20,550 And for fatty acid biosynthesis, the two monomer units 701 00:46:20,550 --> 00:46:22,090 are shown here. 702 00:46:22,090 --> 00:46:26,880 So we have a starter and an extender, acetyl CoA or malonyl 703 00:46:26,880 --> 00:46:29,070 CoA here. 704 00:46:29,070 --> 00:46:31,620 And here we have coenzyme A. 705 00:46:31,620 --> 00:46:35,665 So just as a brief review, if we think about these monomer 706 00:46:35,665 --> 00:46:36,165 units-- 707 00:47:01,730 --> 00:47:07,862 so here we have acetyl CoA. 708 00:47:14,620 --> 00:47:28,680 So what can we say about this guy here, in this thioester? 709 00:47:45,950 --> 00:47:50,280 So is this acidic or not? 710 00:47:57,400 --> 00:47:59,090 Compared to an oxoester. 711 00:48:04,000 --> 00:48:06,890 How many of you have heard about fatty acid biosynthesis? 712 00:48:10,744 --> 00:48:13,700 AUDIENCE: [INAUDIBLE] 713 00:48:13,700 --> 00:48:17,230 ELIZABETH NOLAN: So why are thioesters used and not 714 00:48:17,230 --> 00:48:17,769 oxoesters? 715 00:48:17,769 --> 00:48:19,436 AUDIENCE: [INAUDIBLE] use the other end? 716 00:48:25,497 --> 00:48:26,330 ELIZABETH NOLAN: OK. 717 00:48:26,330 --> 00:48:30,260 So we'll go into a little more detail on Friday 718 00:48:30,260 --> 00:48:32,840 to make sure the chemistry is straight here 719 00:48:32,840 --> 00:48:34,700 because I'm not certain it is. 720 00:48:34,700 --> 00:48:36,188 So-- 721 00:48:36,188 --> 00:48:42,447 AUDIENCE: Is oxoester referring to not that [INAUDIBLE]---- 722 00:48:42,447 --> 00:48:43,280 ELIZABETH NOLAN: OK. 723 00:48:43,280 --> 00:49:02,710 So for Friday, think about a thioester versus an oxoester, 724 00:49:02,710 --> 00:49:05,080 and how do properties differ? 725 00:49:05,080 --> 00:49:09,970 And why might we want to be using thioesters? 726 00:49:09,970 --> 00:49:12,340 And also review the Claisen condensation 727 00:49:12,340 --> 00:49:14,320 because that's the chemistry that's 728 00:49:14,320 --> 00:49:17,230 going to be happening to form the carbon-carbon bonds 729 00:49:17,230 --> 00:49:22,270 in the fatty acid synthase and in the polyketide synthases. 730 00:49:22,270 --> 00:49:25,510 And what we're going to see is that the monomers in each case, 731 00:49:25,510 --> 00:49:26,860 they're tethered as thioesters. 732 00:49:26,860 --> 00:49:28,450 So why is that? 733 00:49:33,750 --> 00:49:38,450 And I will turn around and point at somebody, 734 00:49:38,450 --> 00:49:40,320 and you can let us know. 735 00:49:40,320 --> 00:49:42,540 Are you excited? 736 00:49:42,540 --> 00:49:43,110 OK. 737 00:49:43,110 --> 00:49:46,140 So you're off the hook for Wednesday. 738 00:49:46,140 --> 00:49:49,970 I need to be out of town, and I'll see you on Friday.