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 --> 00:00:18,480 at ocw.mit.edu. 8 00:00:25,847 --> 00:00:27,430 PROFESSOR: We're going to get started. 9 00:00:27,430 --> 00:00:31,390 And today we're going to move forward with the protein 10 00:00:31,390 --> 00:00:35,410 degradation module, and looking at the degradation 11 00:00:35,410 --> 00:00:37,900 chamber of E. coli, ClpXP. 12 00:00:37,900 --> 00:00:41,296 So this is a degradation machine. 13 00:00:41,296 --> 00:00:45,610 And effectively, what we see in this case, 14 00:00:45,610 --> 00:00:47,800 and as mentioned in lecture last time, 15 00:00:47,800 --> 00:00:51,370 is that there are gigantic chambers that 16 00:00:51,370 --> 00:00:54,460 isolate protease active sites. 17 00:00:54,460 --> 00:00:57,040 And so we're going to examine this particular machinery 18 00:00:57,040 --> 00:00:59,320 as a paradigm here. 19 00:00:59,320 --> 00:01:02,710 So the Clp system was first identified in E. coli, 20 00:01:02,710 --> 00:01:05,373 and it's highly conserved. 21 00:01:05,373 --> 00:01:06,790 And what we'll see is that there's 22 00:01:06,790 --> 00:01:11,140 encapsulation of an active site in a large degradation chamber. 23 00:01:11,140 --> 00:01:14,110 So there's two components, ClpX and ClpP. 24 00:01:14,110 --> 00:01:16,360 And so we're going to look at both of these components 25 00:01:16,360 --> 00:01:19,960 individually, and then see how this machine works. 26 00:01:19,960 --> 00:01:26,560 And so ClpP is the proteasome. 27 00:01:32,260 --> 00:01:33,420 And it's a serine protease. 28 00:01:36,520 --> 00:01:39,940 So we talked about serine proteases last time. 29 00:01:39,940 --> 00:01:44,910 So there's the catalytic triad of serine, histidine, 30 00:01:44,910 --> 00:01:46,120 and aspartate. 31 00:01:46,120 --> 00:01:48,790 So there's formation of a covalent 32 00:01:48,790 --> 00:01:50,140 acyl-enzyme intermediate. 33 00:01:50,140 --> 00:01:52,390 We learned that the serine residue is the active site 34 00:01:52,390 --> 00:01:53,680 nucleophile. 35 00:01:53,680 --> 00:01:57,070 And what we're going to see is that this degradation chamber 36 00:01:57,070 --> 00:01:58,540 has 14 active sites. 37 00:02:07,640 --> 00:02:17,900 And what this does is degrade proteins 38 00:02:17,900 --> 00:02:21,290 into short polypeptides. 39 00:02:21,290 --> 00:02:28,970 So those are peptides of about seven to eight amino acids. 40 00:02:33,300 --> 00:02:37,800 And so if we consider a cartoon of the structure, what we find 41 00:02:37,800 --> 00:02:46,720 is that we have two back-to-back rings here. 42 00:02:46,720 --> 00:02:49,390 And each of these rings is a 7-mer. 43 00:02:49,390 --> 00:02:50,650 So we have two heptamers. 44 00:02:57,150 --> 00:03:01,470 In terms of size, they're approximately 90 angstroms 45 00:03:01,470 --> 00:03:08,220 here and approximately 90 angstroms across. 46 00:03:08,220 --> 00:03:10,260 And then this region between the two rings 47 00:03:10,260 --> 00:03:12,015 is sometimes referred to as the equator. 48 00:03:17,030 --> 00:03:18,860 So here we have two back-to-back rings. 49 00:03:26,410 --> 00:03:28,195 And these rings generate a chamber. 50 00:03:36,350 --> 00:03:44,060 And proteins upwards of about 70 kilodaltons can fit. 51 00:03:51,180 --> 00:03:54,100 And so if we take this, looking at from the side, 52 00:03:54,100 --> 00:03:56,400 and just rotate 90 degrees to ask, what does it 53 00:03:56,400 --> 00:03:59,250 look like from the top? 54 00:03:59,250 --> 00:04:00,450 So this is a side view. 55 00:04:25,880 --> 00:04:26,380 OK. 56 00:04:26,380 --> 00:04:32,320 What we see is that there is a very small pore here. 57 00:04:32,320 --> 00:04:34,540 So we have the seven subunits. 58 00:04:34,540 --> 00:04:38,590 And then in the center there is an axial pore. 59 00:04:42,160 --> 00:04:46,445 And this pore is small, about 10 angstroms in diameter. 60 00:04:55,240 --> 00:04:56,890 So when thinking about that size, 61 00:04:56,890 --> 00:05:00,160 we need to think about the size of some large protein, right? 62 00:05:00,160 --> 00:05:03,190 If we have something on the order of 70 kilodaltons 63 00:05:03,190 --> 00:05:05,710 with a fold, that protein's not going 64 00:05:05,710 --> 00:05:09,430 to fit through this hole in that state here. 65 00:05:09,430 --> 00:05:10,600 OK? 66 00:05:10,600 --> 00:05:13,720 So it's too small for a big, folded protein. 67 00:05:13,720 --> 00:05:17,440 But then basically, if we take this and, rather than just 68 00:05:17,440 --> 00:05:19,300 looking at the top, we cut through 69 00:05:19,300 --> 00:05:22,390 and ask what's going on in the interior, what do we see? 70 00:05:25,840 --> 00:05:27,220 OK, so cut through. 71 00:05:39,550 --> 00:05:44,200 What we see now is that there's a chamber 72 00:05:44,200 --> 00:05:45,790 of about 51 angstroms. 73 00:05:49,030 --> 00:05:55,000 OK, so this is the interior degradation chamber. 74 00:06:02,850 --> 00:06:05,640 OK, so one question we're going to address as we move forward 75 00:06:05,640 --> 00:06:08,490 is that, how is it that a polypeptide gets 76 00:06:08,490 --> 00:06:11,370 through this hole into the degradation chamber that 77 00:06:11,370 --> 00:06:15,870 can accommodate a protein up to 17 kilodaltons? 78 00:06:15,870 --> 00:06:22,080 So small axial pore versus large degradation chamber here. 79 00:06:24,660 --> 00:06:30,630 So we'll look at some structures of ClpP and then go on to ClpX. 80 00:06:30,630 --> 00:06:34,500 So what we're looking at here are effectively 81 00:06:34,500 --> 00:06:37,350 what I've drawn out in cartoon form on the board. 82 00:06:37,350 --> 00:06:40,650 So here we have the side view of ClpP. 83 00:06:40,650 --> 00:06:44,100 We have the top ring, the bottom ring, and here's 84 00:06:44,100 --> 00:06:46,650 the region between the two, the equator. 85 00:06:46,650 --> 00:06:48,780 If we look at the top, here they're 86 00:06:48,780 --> 00:06:53,010 describing the axial pore as a portal. 87 00:06:53,010 --> 00:06:54,360 And here's the cutaway view. 88 00:06:54,360 --> 00:06:56,250 So this hole is very small. 89 00:06:56,250 --> 00:06:58,710 And if we look at the cutaway view, what we see 90 00:06:58,710 --> 00:07:02,130 is the degradation chamber here. 91 00:07:02,130 --> 00:07:04,740 And basically, the seven different serine 92 00:07:04,740 --> 00:07:09,510 protease active sites are shown here. 93 00:07:09,510 --> 00:07:13,320 And this is a side view cutting through the middle here. 94 00:07:13,320 --> 00:07:17,490 So we see that these serine protease active sites are 95 00:07:17,490 --> 00:07:21,510 down in this region here. 96 00:07:21,510 --> 00:07:25,380 If we take another view and look at-- again, this is cutaway, 97 00:07:25,380 --> 00:07:28,500 so cut through the side view-- 98 00:07:28,500 --> 00:07:32,070 what we can look at here is the machinery in the active site. 99 00:07:32,070 --> 00:07:36,480 So we learned last time about the catalytic triad, 100 00:07:36,480 --> 00:07:39,330 with the aspartate, histidine, and serine. 101 00:07:39,330 --> 00:07:41,880 And in this particular structure there's 102 00:07:41,880 --> 00:07:46,530 a serine protease inhibitor bound to the serine side chain 103 00:07:46,530 --> 00:07:47,700 here. 104 00:07:47,700 --> 00:07:51,030 So that can serve machinery. 105 00:07:51,030 --> 00:07:54,600 If we look at the structure of an individual ClpP subunit, 106 00:07:54,600 --> 00:07:59,250 those are shown here from several different organisms. 107 00:07:59,250 --> 00:08:02,880 What we see-- if we can think about this as the top part, 108 00:08:02,880 --> 00:08:05,280 and this is the bottom part of one ring-- 109 00:08:05,280 --> 00:08:08,550 we see there's a region with axial loops. 110 00:08:08,550 --> 00:08:12,930 There's a head domain and what's called a handle region. 111 00:08:12,930 --> 00:08:16,710 And the catalytic triad is located at the juncture 112 00:08:16,710 --> 00:08:18,690 of the head and handle region. 113 00:08:18,690 --> 00:08:21,600 And you can, again, look back to the cutaway views 114 00:08:21,600 --> 00:08:24,690 to orient that within the whole chamber. 115 00:08:24,690 --> 00:08:28,020 These axial loops, we'll see, are important for interaction 116 00:08:28,020 --> 00:08:29,580 with ClpX. 117 00:08:29,580 --> 00:08:31,950 And we'll talk about that component 118 00:08:31,950 --> 00:08:34,630 of the machine in a moment. 119 00:08:34,630 --> 00:08:39,389 Here, again, just structures of ClpP from various organisms, 120 00:08:39,389 --> 00:08:42,510 E. coli, Streptococcus, human. 121 00:08:42,510 --> 00:08:47,620 We see that they're all very similar here. 122 00:08:47,620 --> 00:08:50,780 So what is ClpX? 123 00:08:50,780 --> 00:08:51,405 Moving forward. 124 00:09:01,740 --> 00:09:02,250 OK. 125 00:09:02,250 --> 00:09:06,510 So ClpX is effectively an accessory protein. 126 00:09:11,680 --> 00:09:13,480 And in some respects we can think about it 127 00:09:13,480 --> 00:09:15,370 as a lid to the proteasome. 128 00:09:21,580 --> 00:09:22,080 OK. 129 00:09:22,080 --> 00:09:22,705 It's a hexamer. 130 00:09:27,910 --> 00:09:31,570 So there's a mismatch here, in terms of the number of subunits 131 00:09:31,570 --> 00:09:32,870 in ClpP and ClpX. 132 00:09:32,870 --> 00:09:35,670 This is different from what we saw with GroEL, GroES, 133 00:09:35,670 --> 00:09:39,360 where they are both heptamers. 134 00:09:39,360 --> 00:09:41,100 ClpX is a hexamer. 135 00:09:41,100 --> 00:09:43,880 And it's an AAA-- 136 00:09:43,880 --> 00:09:46,700 so triple-A-plus unfoldase. 137 00:09:51,240 --> 00:09:56,630 It's an ATPase here. 138 00:09:56,630 --> 00:09:59,250 And effectively, what we'll see is 139 00:09:59,250 --> 00:10:03,810 that ClpX has an important role as an accessory protein that 140 00:10:03,810 --> 00:10:06,630 unfolds the polypeptide that's destined 141 00:10:06,630 --> 00:10:08,460 for degradation by ClpP. 142 00:10:19,560 --> 00:10:21,450 So it unfolds the polypeptide, and we're 143 00:10:21,450 --> 00:10:23,760 going to have to ask how as we go through. 144 00:10:32,700 --> 00:10:33,220 OK. 145 00:10:33,220 --> 00:10:35,880 And in addition to unfolding, it also 146 00:10:35,880 --> 00:10:39,180 threads that polypeptide that's being unfolded in 147 00:10:39,180 --> 00:10:40,890 through the axial pores such that it 148 00:10:40,890 --> 00:10:43,980 can reach the degradation chamber, 149 00:10:43,980 --> 00:10:56,235 and threads it into the degradation chamber. 150 00:11:02,500 --> 00:11:13,720 And so if we look at ClpX from a top view, 151 00:11:13,720 --> 00:11:14,780 again, we have a hole. 152 00:11:25,230 --> 00:11:33,120 And we have a 6-mer here. 153 00:11:33,120 --> 00:11:40,200 So if we take a look, in this particular depiction what 154 00:11:40,200 --> 00:11:44,680 we're seeing are the top views and the side views. 155 00:11:44,680 --> 00:11:46,560 So here we have ClpP. 156 00:11:46,560 --> 00:11:48,450 Here we have ClpX. 157 00:11:48,450 --> 00:11:53,190 And what we want to ask is, how is it that ClpX binds to ClpP? 158 00:11:53,190 --> 00:11:56,290 And how is this mismatch in terms of the number of subunits 159 00:11:56,290 --> 00:11:57,450 accommodated, right? 160 00:11:57,450 --> 00:11:59,700 So it's not that one subunit is precisely 161 00:11:59,700 --> 00:12:02,260 going to act with one, because we have six in-- 162 00:12:02,260 --> 00:12:02,760 throughout. 163 00:12:02,760 --> 00:12:06,720 Because we have six in ClpX and seven in ClpP. 164 00:12:06,720 --> 00:12:24,020 And so if we think about how ClpX binds to ClpP, what we see 165 00:12:24,020 --> 00:12:27,530 is that we have the 6-mer. 166 00:12:27,530 --> 00:12:29,180 So we're looking from the side view. 167 00:12:31,790 --> 00:12:35,615 And there's some loops that are called IGF loops. 168 00:12:38,840 --> 00:12:40,610 So these are tripeptide motifs. 169 00:12:43,990 --> 00:12:44,865 And they're flexible. 170 00:12:49,250 --> 00:12:54,740 And these loops interact with hydrophobic regions of ClpP. 171 00:13:11,330 --> 00:13:14,660 And this flexibility helps accommodate 172 00:13:14,660 --> 00:13:17,000 the six versus seven subunits. 173 00:13:17,000 --> 00:13:23,240 So if we look here, here we see these tripeptide loops. 174 00:13:23,240 --> 00:13:25,190 And see, we're only seeing three. 175 00:13:25,190 --> 00:13:30,350 But there's one per subunit, so 1, 2, 3, 4, 5, 6 here. 176 00:13:30,350 --> 00:13:33,020 And then what's shown here in red 177 00:13:33,020 --> 00:13:37,050 are the hydrophobic regions of ClpP where these can bind. 178 00:13:39,770 --> 00:13:44,060 And where do we see these regions on ClpP here? 179 00:13:44,060 --> 00:13:49,190 They're a bit removed from the axial pore here. 180 00:13:49,190 --> 00:13:52,700 So how many of these loops are needed? 181 00:13:52,700 --> 00:13:56,150 Just to note, there's been studies done where 182 00:13:56,150 --> 00:13:57,470 these residues are deleted. 183 00:13:57,470 --> 00:14:00,440 And the question is, how many of these motifs 184 00:14:00,440 --> 00:14:05,210 are important for this protein-protein interaction? 185 00:14:05,210 --> 00:14:07,890 And what's been found in test-tube studies 186 00:14:07,890 --> 00:14:11,150 is that a minimum of two are required to get interaction 187 00:14:11,150 --> 00:14:15,676 between ClpX and ClpP here. 188 00:14:15,676 --> 00:14:16,176 Yeah. 189 00:14:16,176 --> 00:14:20,960 AUDIENCE: Is it known how many actually interact in vivo? 190 00:14:20,960 --> 00:14:25,280 Like, do all six interact at any given time? 191 00:14:25,280 --> 00:14:28,080 PROFESSOR: I would presume so, but I don't know. 192 00:14:28,080 --> 00:14:28,580 Right? 193 00:14:28,580 --> 00:14:36,800 So we very much think of this as coming together as shown there. 194 00:14:36,800 --> 00:14:37,800 But don't know. 195 00:14:37,800 --> 00:14:38,820 Joanne, do you know? 196 00:14:38,820 --> 00:14:39,320 No. 197 00:14:39,320 --> 00:14:41,830 No. 198 00:14:41,830 --> 00:14:45,820 I would say it needs to be pretty stable. 199 00:14:45,820 --> 00:14:47,120 Like, there's always dynamics. 200 00:14:47,120 --> 00:14:50,290 But as we see how this machine works, 201 00:14:50,290 --> 00:14:53,380 this hole is going to have to allow the polypeptide to thread 202 00:14:53,380 --> 00:14:57,820 through and get through that axial pore for the polypeptide 203 00:14:57,820 --> 00:14:59,300 to get in the degradation chamber. 204 00:14:59,300 --> 00:15:01,240 So you'd imagine you want that to be lined up 205 00:15:01,240 --> 00:15:05,470 quite well in order for it to be efficient there. 206 00:15:05,470 --> 00:15:06,400 OK. 207 00:15:06,400 --> 00:15:10,270 So what are these triple-A-plus ATPases? 208 00:15:13,330 --> 00:15:18,620 This is a very important group of ATPases. 209 00:15:18,620 --> 00:15:22,180 So what the name means, ATPases associated 210 00:15:22,180 --> 00:15:24,670 with various cellular activities. 211 00:15:24,670 --> 00:15:27,250 And they're super-duper, given this. 212 00:15:27,250 --> 00:15:29,320 Hopefully everyone gets a triple-A-plus 213 00:15:29,320 --> 00:15:31,790 on the exam tonight. 214 00:15:31,790 --> 00:15:36,550 The superfamily is involved in many cellular functions, and-- 215 00:15:36,550 --> 00:15:37,310 take a look. 216 00:15:37,310 --> 00:15:39,150 So, many diverse functions. 217 00:15:39,150 --> 00:15:42,010 Cell membrane fusion, trafficking 218 00:15:42,010 --> 00:15:46,090 of vesicles, cytoskeleton regulation, transport, 219 00:15:46,090 --> 00:15:48,790 organelle biogenesis, DNA replication, 220 00:15:48,790 --> 00:15:50,670 transcription regulation. 221 00:15:50,670 --> 00:15:54,230 And what we're really interested in here is protein degradation. 222 00:15:54,230 --> 00:15:58,780 So they come up in a variety of processes. 223 00:15:58,780 --> 00:16:02,470 And although these processes are very different, 224 00:16:02,470 --> 00:16:04,810 all of these triple-A-plus ATPases 225 00:16:04,810 --> 00:16:08,530 share a common protein architecture. 226 00:16:08,530 --> 00:16:13,260 And I'll just point out that there's an ATP binding module. 227 00:16:13,260 --> 00:16:16,360 And some details are given here in terms of the motifs. 228 00:16:16,360 --> 00:16:19,060 And then really what we'll focus on, 229 00:16:19,060 --> 00:16:20,800 in terms of aspects of this course, 230 00:16:20,800 --> 00:16:26,230 is that they form oligomers that are ring- or cylinder-shaped. 231 00:16:26,230 --> 00:16:30,130 And they're all hexamers here. 232 00:16:30,130 --> 00:16:35,410 And so, of importance to ClpXP, these ATPases 233 00:16:35,410 --> 00:16:38,980 have the ability to remodel conformation of macromolecules. 234 00:16:38,980 --> 00:16:40,682 And so here we're focused on unfolding. 235 00:16:40,682 --> 00:16:41,182 Yeah. 236 00:16:41,182 --> 00:16:42,849 AUDIENCE: [INAUDIBLE] question, but what 237 00:16:42,849 --> 00:16:44,790 are ATPases that aren't associated 238 00:16:44,790 --> 00:16:46,090 with cellular activities? 239 00:16:48,650 --> 00:16:50,480 PROFESSOR: Well-- [LAUGHS] [INAUDIBLE] 240 00:16:50,480 --> 00:16:55,270 AUDIENCE: Is this definition based on the architecture? 241 00:16:55,270 --> 00:16:56,490 PROFESSOR: Strictly, yeah. 242 00:16:56,490 --> 00:16:57,198 I mean, there's-- 243 00:16:57,198 --> 00:17:00,060 GUEST SPEAKER: If you look at tRNA synthetases, 244 00:17:00,060 --> 00:17:01,550 they have ATPase activity. 245 00:17:01,550 --> 00:17:03,992 Hundreds of proteins have ATPase activities. 246 00:17:03,992 --> 00:17:04,700 PROFESSOR: Right. 247 00:17:04,700 --> 00:17:05,260 So-- 248 00:17:05,260 --> 00:17:07,430 GUEST SPEAKER: They hydrolyze ATP to ADP and Pi. 249 00:17:07,430 --> 00:17:08,680 AUDIENCE: Right, right, right. 250 00:17:08,680 --> 00:17:10,770 But that's a cellular activity, right? 251 00:17:10,770 --> 00:17:17,420 AUDIENCE: So, like, what aren't AAA-plus ATPases? 252 00:17:17,420 --> 00:17:20,250 PROFESSOR: Well, aminoacyl-tRNA synthetases 253 00:17:20,250 --> 00:17:23,300 are not triple-A-plus ATPase. 254 00:17:23,300 --> 00:17:25,670 What we'll see in terms of the non-ribosomal peptide 255 00:17:25,670 --> 00:17:29,220 synthetases, they're not these triple-A-plus ATPases. 256 00:17:29,220 --> 00:17:29,720 Right? 257 00:17:29,720 --> 00:17:31,580 So these-- I mean, yes, OK. 258 00:17:31,580 --> 00:17:33,860 All ATPases, the enzymes in a cell, 259 00:17:33,860 --> 00:17:37,310 it's going to have some role in a cellular activity, right? 260 00:17:37,310 --> 00:17:39,830 So maybe this name isn't very helpful. 261 00:17:39,830 --> 00:17:41,810 But what's common about all of these 262 00:17:41,810 --> 00:17:44,300 is that they share this common structural motif. 263 00:17:44,300 --> 00:17:46,057 They form these hexamers. 264 00:17:46,057 --> 00:17:48,140 But within that, there's quite a bit of diversity, 265 00:17:48,140 --> 00:17:50,140 because they have all these different functions. 266 00:17:50,140 --> 00:17:53,480 So we can just see that here, to some degree. 267 00:17:53,480 --> 00:17:54,760 So-- oops. 268 00:17:54,760 --> 00:17:57,680 And there's a typo, which I'll fix before posting. 269 00:17:57,680 --> 00:18:02,030 But if we take a look just at two examples here of different 270 00:18:02,030 --> 00:18:05,300 hexameric rings-- so these are two different triple-A-plus 271 00:18:05,300 --> 00:18:07,370 ATPases-- 272 00:18:07,370 --> 00:18:08,660 what do we see? 273 00:18:08,660 --> 00:18:12,170 So in common, they're both hexamers. 274 00:18:12,170 --> 00:18:17,750 In common, they both have an axial pore here. 275 00:18:17,750 --> 00:18:21,440 But we see different elements of secondary structure. 276 00:18:21,440 --> 00:18:22,973 And granted, these are both depicted 277 00:18:22,973 --> 00:18:24,140 in a bit of a different way. 278 00:18:24,140 --> 00:18:25,548 But if we look here-- 279 00:18:25,548 --> 00:18:26,090 I mean, look. 280 00:18:26,090 --> 00:18:29,900 We have these alpha helical regions around the exterior 281 00:18:29,900 --> 00:18:32,750 that we don't see here. 282 00:18:32,750 --> 00:18:34,100 And in this view-- 283 00:18:34,100 --> 00:18:36,860 I show this particular one because it's depicted 284 00:18:36,860 --> 00:18:38,820 where the ATP is binding. 285 00:18:38,820 --> 00:18:41,660 So you can see the ATP binding to each subunit here. 286 00:18:41,660 --> 00:18:46,400 So, as shown, six ATPs bound. 287 00:18:46,400 --> 00:18:49,290 So the structural diversity is quite tremendous. 288 00:18:49,290 --> 00:18:51,150 And here's just another example. 289 00:18:51,150 --> 00:18:55,310 So these are three different triple-A-plus ATPases 290 00:18:55,310 --> 00:18:56,390 of the Clp system. 291 00:18:56,390 --> 00:19:01,080 So we're going to focus on ClpX, but it's not the only one. 292 00:19:01,080 --> 00:19:04,190 And so what we're looking at here is ClpX. 293 00:19:04,190 --> 00:19:09,140 We have another family member, ClpA, and here, ClpB. 294 00:19:09,140 --> 00:19:12,080 And so what we see is, subunit to subunit, 295 00:19:12,080 --> 00:19:16,110 whether it's X, A, or B, there's quite a bit of difference, 296 00:19:16,110 --> 00:19:16,610 right? 297 00:19:16,610 --> 00:19:20,390 So ClpX is the most simple, in terms of the architecture here, 298 00:19:20,390 --> 00:19:22,450 for that. 299 00:19:22,450 --> 00:19:24,680 And so one thing people think about is, 300 00:19:24,680 --> 00:19:26,300 in terms of the different activities 301 00:19:26,300 --> 00:19:29,150 that have been associated with these different family members, 302 00:19:29,150 --> 00:19:31,510 how is it that these different structural features play 303 00:19:31,510 --> 00:19:32,366 a role? 304 00:19:32,366 --> 00:19:33,340 OK. 305 00:19:33,340 --> 00:19:35,120 Here. 306 00:19:35,120 --> 00:19:45,170 So, coming back to ClpX and the depiction we saw before, 307 00:19:45,170 --> 00:19:48,140 ClpX is an unfoldase. 308 00:19:48,140 --> 00:19:50,930 And what's really a key point here 309 00:19:50,930 --> 00:19:55,640 is that ATP hydrolysis by ClpX is 310 00:19:55,640 --> 00:19:58,610 going to power conformational changes that 311 00:19:58,610 --> 00:20:03,080 allow for mechanical unfolding of this protein that's 312 00:20:03,080 --> 00:20:07,130 condemned for degradation by ClpP. 313 00:20:07,130 --> 00:20:10,100 And that's what's going to also allow for translocation 314 00:20:10,100 --> 00:20:12,740 of the resulting unfolded protein 315 00:20:12,740 --> 00:20:14,780 into the degradation chamber. 316 00:20:14,780 --> 00:20:17,690 So the action of ClpX is allowing that protein 317 00:20:17,690 --> 00:20:20,120 to fit through this axial pore and be 318 00:20:20,120 --> 00:20:21,590 threaded into the chamber. 319 00:20:24,470 --> 00:20:27,110 So with that, what are the questions 320 00:20:27,110 --> 00:20:30,980 we need to address in thinking about how this macromolecular 321 00:20:30,980 --> 00:20:33,200 machine works? 322 00:20:33,200 --> 00:20:36,290 One, how are substrates recognized? 323 00:20:36,290 --> 00:20:38,510 So there's some certain group of proteins 324 00:20:38,510 --> 00:20:41,570 that are going to be degraded by this machinery. 325 00:20:41,570 --> 00:20:43,580 What is the mechanism? 326 00:20:43,580 --> 00:20:47,210 How is it that ATP-dependent conformational changes 327 00:20:47,210 --> 00:20:51,410 of ClpXP drive unfolding and translocation? 328 00:20:51,410 --> 00:20:53,480 And what is the substrate selectivity? 329 00:20:53,480 --> 00:20:56,940 So that's where we're going to move forward with. 330 00:20:56,940 --> 00:20:59,480 And so the first question we need to ask 331 00:20:59,480 --> 00:21:03,450 is, how are the substrates recognized by ClpX? 332 00:21:03,450 --> 00:21:04,300 OK? 333 00:21:04,300 --> 00:21:04,800 Here. 334 00:21:18,310 --> 00:21:20,420 And so, what are possibilities? 335 00:21:27,620 --> 00:21:28,430 OK. 336 00:21:28,430 --> 00:21:32,210 First, what we'll consider is a degradation tag. 337 00:21:39,150 --> 00:21:41,040 So when I draw these cartoons, I'm 338 00:21:41,040 --> 00:21:44,643 only going to show one of the two rings for ClpP. 339 00:21:44,643 --> 00:21:45,810 It's not that it's only one. 340 00:21:45,810 --> 00:21:47,730 This is just for simplicity. 341 00:21:47,730 --> 00:22:02,970 But imagine that here we have X, here we have P. 342 00:22:02,970 --> 00:22:05,610 And we have some condemned protein, which 343 00:22:05,610 --> 00:22:06,735 I'll just draw as a circle. 344 00:22:10,450 --> 00:22:12,820 So the cell no longer wants this protein. 345 00:22:12,820 --> 00:22:13,870 It needs to go away. 346 00:22:16,780 --> 00:22:19,750 And we can imagine, as one possibility, 347 00:22:19,750 --> 00:22:25,600 is that a degradation tag can be attached to this polypeptide. 348 00:22:25,600 --> 00:22:28,870 And what we find is that there's a particular tag called 349 00:22:28,870 --> 00:22:36,100 ssrA tag that is used to tag proteins for degradation 350 00:22:36,100 --> 00:22:38,170 by ClpXP. 351 00:22:38,170 --> 00:22:40,510 So we can think of this tag as a zip code. 352 00:22:43,090 --> 00:22:47,650 If a polypeptide gets modified such that this tag is appended, 353 00:22:47,650 --> 00:22:51,790 it's going to end up going to this degradation machine such 354 00:22:51,790 --> 00:22:53,680 that it gets degraded. 355 00:22:53,680 --> 00:22:55,675 The tag is 11 amino acids. 356 00:23:00,870 --> 00:23:02,700 It's attached to the C-terminus. 357 00:23:11,360 --> 00:23:11,880 OK. 358 00:23:11,880 --> 00:23:20,641 And the sequence is A, A, N, D, E, N, Y, A, L, A, 359 00:23:20,641 --> 00:23:29,100 A. And so what happens in this case, as shown-- 360 00:23:29,100 --> 00:23:35,550 we can imagine that this tag binds to the pore of ClpX 361 00:23:35,550 --> 00:23:36,840 directly. 362 00:23:36,840 --> 00:23:42,330 And the tag, when binding, enables translocation. 363 00:23:42,330 --> 00:23:42,900 So here-- 364 00:24:08,680 --> 00:24:09,180 OK. 365 00:24:09,180 --> 00:24:18,500 And this pore has what are termed pore loops that 366 00:24:18,500 --> 00:24:19,865 are involved in tag binding. 367 00:24:27,400 --> 00:24:33,560 And in particular, there's a region, GYVG-- 368 00:24:33,560 --> 00:24:36,280 so, a four-amino-acid sequence-- 369 00:24:36,280 --> 00:24:44,090 that is thought to grip and drag the substrate. 370 00:24:47,866 --> 00:24:48,790 OK? 371 00:24:48,790 --> 00:24:49,290 Here. 372 00:24:49,290 --> 00:24:51,480 And of course it's not gripping like we would, 373 00:24:51,480 --> 00:24:53,970 but there's some interaction there happening 374 00:24:53,970 --> 00:24:55,370 that allows that to occur. 375 00:24:55,370 --> 00:24:58,800 So you'll see there's a lot of mechanical-type cartoons 376 00:24:58,800 --> 00:25:02,860 and language used in describing these machines. 377 00:25:02,860 --> 00:25:03,360 OK. 378 00:25:03,360 --> 00:25:05,340 So what is another possibility? 379 00:25:21,420 --> 00:25:25,590 So another possibility for how an ATPase could interact 380 00:25:25,590 --> 00:25:39,180 with a degradation chamber is that the protein substrate 381 00:25:39,180 --> 00:25:49,810 binds to an extra domain attached to the ATPase. 382 00:25:59,590 --> 00:26:00,090 OK. 383 00:26:00,090 --> 00:26:03,480 And I point out, this possibility is not for ClpX, 384 00:26:03,480 --> 00:26:05,850 but it's one to be aware of, because it can occur. 385 00:26:12,680 --> 00:26:16,500 We saw some of those other ATPase are quite complicated. 386 00:26:16,500 --> 00:26:23,870 So in this case, imagine we have our ATPase, 387 00:26:23,870 --> 00:26:27,830 we have the degradation chamber. 388 00:26:27,830 --> 00:26:41,310 And this ATPase has some extra domain 389 00:26:41,310 --> 00:26:52,800 that effectively can bind the condemned protein 390 00:26:52,800 --> 00:26:56,490 and help deliver it to the pore here. 391 00:26:59,480 --> 00:27:02,320 And just as a third possibility, and something 392 00:27:02,320 --> 00:27:04,360 that we'll see moving forward, is 393 00:27:04,360 --> 00:27:07,750 that there is involvement of an adaptor protein. 394 00:27:07,750 --> 00:27:11,650 So in addition to the ATPase and the degradation chamber, 395 00:27:11,650 --> 00:27:15,290 there's an adaptor protein that comes into play. 396 00:27:15,290 --> 00:27:16,615 So in this case, the protein-- 397 00:27:29,942 --> 00:27:30,825 OK, adaptor-- 398 00:27:37,420 --> 00:27:37,920 OK. 399 00:27:37,920 --> 00:27:40,795 And this protein helps direct it to the pore-- 400 00:27:48,300 --> 00:27:56,905 so, the condemned protein to the ATPase. 401 00:27:59,300 --> 00:27:59,800 OK. 402 00:27:59,800 --> 00:28:06,093 So for instance, here we have the ATPase. 403 00:28:09,290 --> 00:28:13,460 Here we have the degradation chamber. 404 00:28:13,460 --> 00:28:26,370 And maybe there's some additional protein 405 00:28:26,370 --> 00:28:33,440 that facilitates getting the condemned 406 00:28:33,440 --> 00:28:35,400 protein to the ATPase. 407 00:28:38,060 --> 00:28:40,220 And so something to keep in mind, 408 00:28:40,220 --> 00:28:47,510 and what we'll see with ClpXP, is that one and three are not 409 00:28:47,510 --> 00:28:48,710 mutually exclusive. 410 00:28:56,920 --> 00:28:59,110 And there's an adaptor protein named 411 00:28:59,110 --> 00:29:09,310 SspB that can help deliver ssrA-tagged polypeptides 412 00:29:09,310 --> 00:29:14,410 or proteins to the degradation chamber here. 413 00:29:21,900 --> 00:29:28,550 So we're going to think about these ssrA tags quite a bit. 414 00:29:28,550 --> 00:29:31,040 And something else to be aware of 415 00:29:31,040 --> 00:29:33,320 is just that these ssrA tags are not 416 00:29:33,320 --> 00:29:37,083 the only ways of tagging proteins for degradation. 417 00:29:37,083 --> 00:29:39,500 We're not going to talk about it in detail in this course, 418 00:29:39,500 --> 00:29:41,420 but you should be aware of something 419 00:29:41,420 --> 00:29:44,120 called the N-end rule. 420 00:29:44,120 --> 00:29:45,290 And this is really cool. 421 00:29:45,290 --> 00:29:51,140 So this rule basically states that a half-life of a protein 422 00:29:51,140 --> 00:29:54,290 is determined by its N-terminal residue here. 423 00:29:54,290 --> 00:29:57,110 And this can be called an N-degron. 424 00:29:57,110 --> 00:30:01,640 And these N-degrons are recognized by proteins 425 00:30:01,640 --> 00:30:03,860 such as ClpS and E. coli. 426 00:30:03,860 --> 00:30:08,120 And as a result, these proteins can get delivered 427 00:30:08,120 --> 00:30:09,630 to degradation machines. 428 00:30:09,630 --> 00:30:13,070 So for instance, in addition to ClpXP, 429 00:30:13,070 --> 00:30:17,510 there's an ATPase, ClpA, that can associate with ClpP 430 00:30:17,510 --> 00:30:20,840 and be involved in degradation of polypeptides 431 00:30:20,840 --> 00:30:23,060 via this N-end rule. 432 00:30:23,060 --> 00:30:24,590 And in terms of the rule, depending 433 00:30:24,590 --> 00:30:27,470 on the identity of this N-terminal amino acid, 434 00:30:27,470 --> 00:30:30,530 it may be stabilizing or destabilizing, in terms 435 00:30:30,530 --> 00:30:32,200 of protein lifetime. 436 00:30:32,200 --> 00:30:33,950 If you're curious to know more about that, 437 00:30:33,950 --> 00:30:38,210 we can refer you to some literature. 438 00:30:38,210 --> 00:30:43,820 So here we have a cartoon looking 439 00:30:43,820 --> 00:30:48,470 at a native protein substrate that needs to be degraded. 440 00:30:48,470 --> 00:30:50,570 It's been modified with a tag. 441 00:30:50,570 --> 00:30:52,130 We have ClpX here. 442 00:30:52,130 --> 00:30:54,160 We have ClpP. 443 00:30:54,160 --> 00:30:57,380 Here's the tag. 444 00:30:57,380 --> 00:30:59,890 And in addition, we can have this adaptor protein 445 00:30:59,890 --> 00:31:04,340 SspB and the adaptor ClpS. 446 00:31:04,340 --> 00:31:07,760 So let's think about this tag for a minute. 447 00:31:07,760 --> 00:31:09,320 And we need to think about this tag 448 00:31:09,320 --> 00:31:12,920 from the standpoint, one, of in vitro experiments, 449 00:31:12,920 --> 00:31:15,830 because we're going to begin to look at some experiments that 450 00:31:15,830 --> 00:31:19,220 were done to understand how this machine works. 451 00:31:19,220 --> 00:31:21,770 And we also need to think about this tag 452 00:31:21,770 --> 00:31:25,470 from the standpoint of the cell. 453 00:31:25,470 --> 00:31:29,900 So if we think about an in vitro experiment where 454 00:31:29,900 --> 00:31:34,670 we want to study how ClpXP degrades some protein 455 00:31:34,670 --> 00:31:38,810 substrate, we can use this ssrA tag. 456 00:31:38,810 --> 00:31:43,520 And it's quite easy to attach 11 amino acids to some protein 457 00:31:43,520 --> 00:31:46,670 or polypeptide at the C-terminus. 458 00:31:46,670 --> 00:31:48,470 We can do that by protein expression, 459 00:31:48,470 --> 00:31:52,280 we can do that by chemical synthesis here. 460 00:31:52,280 --> 00:31:55,220 And so we're going to look at a number of experiments 461 00:31:55,220 --> 00:31:58,950 where this ssrA tag has been appended to certain model 462 00:31:58,950 --> 00:32:02,250 substrates, moving forward. 463 00:32:02,250 --> 00:32:04,980 So what about in the cell? 464 00:32:04,980 --> 00:32:08,600 So when is this ssrA tag attached to a protein? 465 00:32:13,250 --> 00:32:19,520 So are all proteins that need to be degraded destined to ClpXP? 466 00:32:19,520 --> 00:32:23,410 Just intuitively, what do you think? 467 00:32:23,410 --> 00:32:24,870 I see shaking heads, no. 468 00:32:24,870 --> 00:32:25,570 Right? 469 00:32:25,570 --> 00:32:29,040 There's many, many proteases around. 470 00:32:29,040 --> 00:32:35,952 So what proteins are destined for degradation by ClpXP? 471 00:32:35,952 --> 00:32:37,410 That's what we're going to look at, 472 00:32:37,410 --> 00:32:41,310 and how this tag is appended. 473 00:32:41,310 --> 00:32:50,610 And so effectively, this ssrA tag, say, in E. coli, is used-- 474 00:32:50,610 --> 00:32:54,960 one, because protein degradation needs to be tightly regulated. 475 00:32:54,960 --> 00:32:59,950 But two, it's used for dealing with proteins that 476 00:32:59,950 --> 00:33:02,530 exhibited stalled translation. 477 00:33:02,530 --> 00:33:06,290 So this discussion is going to bring us back to the ribosome 478 00:33:06,290 --> 00:33:06,790 here. 479 00:33:09,880 --> 00:33:12,220 So we want to ask what proteins in the cell 480 00:33:12,220 --> 00:33:15,340 are tagged with ssrA. 481 00:33:15,340 --> 00:33:19,490 How is the tag attached to the [INAUDIBLE] protein as well 482 00:33:19,490 --> 00:33:21,930 here? 483 00:33:21,930 --> 00:33:24,360 This is just a cartoon showing an adaptor protein 484 00:33:24,360 --> 00:33:31,080 helping direct this tag to the substrate here. 485 00:33:31,080 --> 00:33:36,090 So we're going to just move forward to this slide. 486 00:33:36,090 --> 00:33:39,960 This tag is specifically added to proteins 487 00:33:39,960 --> 00:33:43,710 that are experiencing stalled translation. 488 00:33:43,710 --> 00:33:46,620 So it's estimated that on the order 489 00:33:46,620 --> 00:33:51,960 of 0.5% of E. coli translations result in ssrA tagging. 490 00:33:51,960 --> 00:33:56,220 And so this is thought to be one largely of quality control. 491 00:33:56,220 --> 00:33:59,130 So you can imagine, if the ribosome stalled, 492 00:33:59,130 --> 00:34:02,370 there could be buildup of peptide products 493 00:34:02,370 --> 00:34:04,530 that aren't wanted. 494 00:34:04,530 --> 00:34:06,930 And the translation machinery could get blocked, 495 00:34:06,930 --> 00:34:09,679 and we don't really want that to happen here. 496 00:34:12,199 --> 00:34:16,150 So here's our friend, the ribosome. 497 00:34:16,150 --> 00:34:19,960 And here's looking at the 50S ribosomal subunit. 498 00:34:19,960 --> 00:34:23,050 And we have a polypeptide emerging from the exit tunnel. 499 00:34:23,050 --> 00:34:25,480 So these should all be familiar at this stage. 500 00:34:25,480 --> 00:34:27,520 And so what happens when this ribosome is 501 00:34:27,520 --> 00:34:29,949 trying to synthesize a polypeptide 502 00:34:29,949 --> 00:34:32,020 and it just gets stuck? 503 00:34:32,020 --> 00:34:37,989 So this ssrA tag is attached to the C-terminus of proteins. 504 00:34:37,989 --> 00:34:40,900 And as we're going to see, this occurs cotranslationally. 505 00:34:40,900 --> 00:34:47,120 And it's very, very interesting machinery. 506 00:34:47,120 --> 00:34:51,639 So what we see here is that there's 507 00:34:51,639 --> 00:34:54,400 a new player we haven't yet seen. 508 00:34:54,400 --> 00:34:59,200 And this is called ssrA, or tmRNA, 509 00:34:59,200 --> 00:35:02,470 for transfer messenger RNA. 510 00:35:02,470 --> 00:35:06,130 And it's involved in attachment of this ssrA tag 511 00:35:06,130 --> 00:35:09,960 to polypeptides that are having stalled 512 00:35:09,960 --> 00:35:12,190 biosynthesis on the ribosome. 513 00:35:12,190 --> 00:35:18,130 And so this player acts as both a tRNA and an mRNA. 514 00:35:18,130 --> 00:35:20,840 And we can take a look at the structure shown here. 515 00:35:20,840 --> 00:35:25,180 So here we have tRNA in the cloverleaf depiction, just 516 00:35:25,180 --> 00:35:27,250 an Ala-tRNA Ala. 517 00:35:27,250 --> 00:35:31,000 And if we take a look here, what do we see? 518 00:35:31,000 --> 00:35:34,870 At this end we have a region of the tmRNA 519 00:35:34,870 --> 00:35:37,350 that looks like a tRNA. 520 00:35:37,350 --> 00:35:37,850 Right? 521 00:35:37,850 --> 00:35:42,530 Quite similar here to the [INAUDIBLE] prime end. 522 00:35:42,530 --> 00:35:46,800 And then we have this additional region. 523 00:35:46,800 --> 00:35:50,890 And then if we look down in here, what do we see? 524 00:35:50,890 --> 00:35:53,040 We see a region that, with a little imagination, 525 00:35:53,040 --> 00:35:55,920 we can think looks like mRNA. 526 00:35:55,920 --> 00:36:00,570 And if we take a look at the various codons, what we see 527 00:36:00,570 --> 00:36:08,490 is that the ssrA tag is encoded there, along with a stop codon. 528 00:36:08,490 --> 00:36:11,490 So effectively we have a tRNA look-alike. 529 00:36:11,490 --> 00:36:15,825 We have an mRNA look-alike that is encoding this ssrA tag. 530 00:36:19,830 --> 00:36:22,850 So what happens? 531 00:36:22,850 --> 00:36:28,710 There's a partner protein called smpB just to be aware of. 532 00:36:28,710 --> 00:36:33,690 And in complex with smpB, it's actually 533 00:36:33,690 --> 00:36:42,840 EF-Tu that delivers this tmRNA to the ribosome here. 534 00:36:42,840 --> 00:36:46,320 So this is pretty interesting, just from the standpoint 535 00:36:46,320 --> 00:36:48,540 of what we know about EF-Tu. 536 00:36:48,540 --> 00:36:53,310 We don't have a typical anticodon here. 537 00:36:53,310 --> 00:36:54,622 So how does that happen? 538 00:36:54,622 --> 00:36:56,080 We're not going to go into details, 539 00:36:56,080 --> 00:36:57,780 but it's something-- you know, curiosity 540 00:36:57,780 --> 00:37:01,090 should beg those questions. 541 00:37:01,090 --> 00:37:03,600 So what happens? 542 00:37:03,600 --> 00:37:07,590 We can look at this cartoon overview here. 543 00:37:07,590 --> 00:37:09,750 And so the color coding within this 544 00:37:09,750 --> 00:37:12,930 is helpful, in terms of keeping track of pieces. 545 00:37:12,930 --> 00:37:15,600 But here we start with our stalled ribosome. 546 00:37:15,600 --> 00:37:17,550 So the mRNA is bound. 547 00:37:17,550 --> 00:37:21,310 We see there's a peptidyl tRNA in the P-site. 548 00:37:21,310 --> 00:37:24,780 You know the polypeptide has a number of amino acids, 549 00:37:24,780 --> 00:37:26,640 and the A-site is empty. 550 00:37:26,640 --> 00:37:31,620 And for some reason, no new aminoacyl tRNA is coming in. 551 00:37:31,620 --> 00:37:34,440 So the ribosome stalls. 552 00:37:34,440 --> 00:37:40,140 And as a result, this ssrA, or tmRNA, 553 00:37:40,140 --> 00:37:43,080 is recruited to this stalled ribosome. 554 00:37:43,080 --> 00:37:45,840 And so here we see the tRNA end in yellow, 555 00:37:45,840 --> 00:37:46,935 with the alanine attached. 556 00:37:46,935 --> 00:37:49,980 And here we have that region that's 557 00:37:49,980 --> 00:37:52,650 mRNA-like encoding the tag. 558 00:37:52,650 --> 00:37:55,920 So this biomolecule gets recruited. 559 00:37:55,920 --> 00:37:57,210 And what do we see? 560 00:37:57,210 --> 00:37:59,610 It enters the A-site. 561 00:37:59,610 --> 00:38:03,690 So here we see the tRNA end in A-site, 562 00:38:03,690 --> 00:38:06,900 and we have the rest of the molecule here. 563 00:38:06,900 --> 00:38:08,340 Then what? 564 00:38:08,340 --> 00:38:11,140 There's formation of a new peptide bond, 565 00:38:11,140 --> 00:38:12,780 so we have peptidyl transfer. 566 00:38:12,780 --> 00:38:14,290 We see that alanine is here. 567 00:38:14,290 --> 00:38:14,790 Look. 568 00:38:14,790 --> 00:38:16,665 That looks quite a bit like the hybrid states 569 00:38:16,665 --> 00:38:21,000 we talked about, where we're seeing these ends 570 00:38:21,000 --> 00:38:25,080 shift into the E-site, not shown, and the P-site here. 571 00:38:25,080 --> 00:38:26,340 And then what happens? 572 00:38:26,340 --> 00:38:29,790 There's translocation and there's message switching. 573 00:38:29,790 --> 00:38:33,690 So the original mRNA gets kicked out. 574 00:38:33,690 --> 00:38:35,280 And what do we see? 575 00:38:35,280 --> 00:38:42,970 Now that mRNA-like region of the tRNA is in A-site here. 576 00:38:42,970 --> 00:38:46,480 Then what happens after replacement of the mRNA? 577 00:38:46,480 --> 00:38:48,670 Translation can occur, which results 578 00:38:48,670 --> 00:38:51,760 in synthesis of the ssrA tag. 579 00:38:51,760 --> 00:38:54,910 So that's how this tag is attached to the C-terminus 580 00:38:54,910 --> 00:38:57,300 of the polypeptide. 581 00:38:57,300 --> 00:39:01,240 And elongation occurs until that stop codon in the tmRNA 582 00:39:01,240 --> 00:39:03,250 enters the A-site. 583 00:39:03,250 --> 00:39:06,100 And then peptide release occurs here. 584 00:39:06,100 --> 00:39:09,880 So the result is a protein that has the ssrA 585 00:39:09,880 --> 00:39:12,340 tag attached to its C-terminus. 586 00:39:12,340 --> 00:39:18,070 And that protein will be directed to ClpXP. 587 00:39:18,070 --> 00:39:20,120 So, pretty cool. 588 00:39:20,120 --> 00:39:20,620 Yeah. 589 00:39:20,620 --> 00:39:22,210 I think so. 590 00:39:22,210 --> 00:39:22,840 There. 591 00:39:22,840 --> 00:39:28,300 We don't ever leave the ribosome too much within these units. 592 00:39:28,300 --> 00:39:32,590 Just to point out, this tag is universal in bacteria. 593 00:39:32,590 --> 00:39:36,370 So here's just a table of phylogenetic distribution. 594 00:39:36,370 --> 00:39:40,250 You're not responsible for these details. 595 00:39:40,250 --> 00:39:41,110 Yeah. 596 00:39:41,110 --> 00:39:43,180 AUDIENCE: About the last slide-- 597 00:39:43,180 --> 00:39:47,630 so is the tag attached after it's stalled? 598 00:39:47,630 --> 00:39:49,780 Like, is the original protein completed? 599 00:39:49,780 --> 00:39:55,400 Or just the original mRNA removed and detached the tag? 600 00:39:55,400 --> 00:39:56,313 Or it's both? 601 00:39:56,313 --> 00:39:56,980 PROFESSOR: Yeah. 602 00:39:56,980 --> 00:40:00,380 So what does the cartoon suggest? 603 00:40:00,380 --> 00:40:02,843 AUDIENCE: It feels like it's already on the C-end here. 604 00:40:06,550 --> 00:40:11,620 PROFESSOR: Well, the ribosome can stall at various points. 605 00:40:11,620 --> 00:40:14,830 So imagine you have a 100-amino-acid polypeptide that 606 00:40:14,830 --> 00:40:16,990 needs to be synthesized. 607 00:40:16,990 --> 00:40:22,600 The ribosome could stall after amino acid 20 or 40 or 60. 608 00:40:22,600 --> 00:40:24,280 It's not that the whole polypeptide 609 00:40:24,280 --> 00:40:28,520 has been synthesized and then this gets put on. 610 00:40:28,520 --> 00:40:33,310 It may be some fragment there for that. 611 00:40:33,310 --> 00:40:36,320 So, yes. 612 00:40:39,800 --> 00:40:43,160 So in terms of this adaptor protein, 613 00:40:43,160 --> 00:40:48,080 I just want to make a note in terms of the adaptor. 614 00:40:48,080 --> 00:40:52,220 So these adaptor proteins can help 615 00:40:52,220 --> 00:40:55,700 with regulating the substrate specificity 616 00:40:55,700 --> 00:40:58,970 of triple-A-plus ATPases. 617 00:40:58,970 --> 00:41:03,260 And effectively, depending on the system and depending 618 00:41:03,260 --> 00:41:08,280 on the adaptor, it may enhance or it may inhibit degradation 619 00:41:08,280 --> 00:41:08,780 here. 620 00:41:08,780 --> 00:41:10,220 So it's a case-by-case basis. 621 00:41:12,950 --> 00:41:17,570 This SspB shown in the cartoon here 622 00:41:17,570 --> 00:41:21,800 is a dimeric adaptor for XP, and it promotes degradation 623 00:41:21,800 --> 00:41:23,690 of certain substrates. 624 00:41:23,690 --> 00:41:28,730 And effectively, it enhances recognition of this tag 625 00:41:28,730 --> 00:41:33,380 by the machinery such that the degradation rates are enhanced. 626 00:41:33,380 --> 00:41:35,940 So it's not that it's required. 627 00:41:35,940 --> 00:41:38,210 It's just helpful, and accelerates the process. 628 00:41:58,260 --> 00:42:03,780 So just an interesting observation regarding SspB-- 629 00:42:03,780 --> 00:42:08,400 it can be co-purified with ribosomes here. 630 00:42:08,400 --> 00:42:10,920 And in terms of its structure, it 631 00:42:10,920 --> 00:42:15,570 has some resemblance to known RNA-binding proteins. 632 00:42:15,570 --> 00:42:18,960 And this resemblance has begged a question, 633 00:42:18,960 --> 00:42:24,450 does SspB itself help with linking protein synthesis 634 00:42:24,450 --> 00:42:26,850 and protein degradation? 635 00:42:26,850 --> 00:42:31,770 So is it possible that SspB could 636 00:42:31,770 --> 00:42:37,050 help promote binding of ClpX to polypeptides 637 00:42:37,050 --> 00:42:38,760 before full release to the ribosome? 638 00:42:38,760 --> 00:42:41,100 That's something people have wondered about. 639 00:42:41,100 --> 00:42:44,430 And initially this protein was classified 640 00:42:44,430 --> 00:42:47,040 as a stringent starvation protein. 641 00:42:47,040 --> 00:42:49,270 That's where the name comes from. 642 00:42:49,270 --> 00:42:53,650 So if we just take a quick look at its structure here, 643 00:42:53,650 --> 00:42:55,050 what do we see? 644 00:42:55,050 --> 00:42:57,390 So here is SspB. 645 00:42:57,390 --> 00:43:03,570 And then here we have structures from ribosomal proteins. 646 00:43:03,570 --> 00:43:08,970 And so in SspB, ClpX binds on this side. 647 00:43:08,970 --> 00:43:15,300 And effectively, here we look at the ribosomal proteins 648 00:43:15,300 --> 00:43:16,200 that bind RNA. 649 00:43:16,200 --> 00:43:18,900 And they have these RNA-binding sites there. 650 00:43:18,900 --> 00:43:21,540 So there's some similarities in terms of the alpha helix, 651 00:43:21,540 --> 00:43:23,330 in terms of the beta sheets here. 652 00:43:26,790 --> 00:43:29,100 And also, I'll just note, in terms 653 00:43:29,100 --> 00:43:38,260 of SspB and the ssrA tag-- 654 00:43:38,260 --> 00:43:41,130 so if we take this tag-- 655 00:43:51,230 --> 00:43:55,910 So this is our ssrA tag here. 656 00:43:55,910 --> 00:44:03,350 What's found is that ClpX recognition is on this end 657 00:44:03,350 --> 00:44:06,980 and SspB binding is on this end here. 658 00:44:19,190 --> 00:44:21,050 So in different points. 659 00:44:21,050 --> 00:44:31,790 So what this indicates here is that SspB and ClpX 660 00:44:31,790 --> 00:44:33,410 can bind simultaneously. 661 00:44:41,770 --> 00:44:42,850 OK. 662 00:44:42,850 --> 00:44:45,850 But this is small, so we can expect that there's 663 00:44:45,850 --> 00:44:49,520 some clash here for that. 664 00:44:55,480 --> 00:44:58,000 So where we're going to close is just 665 00:44:58,000 --> 00:45:02,290 looking at an overview as to how this machine works, 666 00:45:02,290 --> 00:45:05,710 and the model that then, starting on Friday, 667 00:45:05,710 --> 00:45:07,750 we'll look at experiments that were designed 668 00:45:07,750 --> 00:45:12,370 and performed to inform this model. 669 00:45:12,370 --> 00:45:16,150 So if we look at this in one type of cartoon, 670 00:45:16,150 --> 00:45:18,740 what are the stages? 671 00:45:18,740 --> 00:45:22,990 So we can think of three as depicted here, 672 00:45:22,990 --> 00:45:25,840 where there's some sort of initial recognition. 673 00:45:25,840 --> 00:45:31,060 So the ssrA tag of this condemned protein 674 00:45:31,060 --> 00:45:34,210 binds to the axial pore of ClpX. 675 00:45:34,210 --> 00:45:39,490 And this process does not require ATP hydrolysis. 676 00:45:39,490 --> 00:45:41,860 So here we see a folded substrate. 677 00:45:41,860 --> 00:45:47,230 This degron is another word for one of these tags, ssrA tag. 678 00:45:47,230 --> 00:45:50,800 So we see there's recognition here. 679 00:45:50,800 --> 00:45:55,100 Then what happens, ClpX unfolds this substrate. 680 00:45:55,100 --> 00:45:57,910 So somehow it has to grip and pull and apply 681 00:45:57,910 --> 00:46:00,580 a force that unfolds the polypeptide, 682 00:46:00,580 --> 00:46:03,370 and threads that unfolded polypeptide 683 00:46:03,370 --> 00:46:04,960 into the degradation chamber. 684 00:46:04,960 --> 00:46:08,330 So, you know, kind of this pulley system is shown here. 685 00:46:08,330 --> 00:46:11,480 This chopper-type thing is shown here. 686 00:46:11,480 --> 00:46:14,500 You can use your imagination in this unit 687 00:46:14,500 --> 00:46:16,630 for how to depict this machine. 688 00:46:16,630 --> 00:46:20,560 So we see that the polypeptide is being unfolded and threaded 689 00:46:20,560 --> 00:46:22,630 through ClpX into this chamber, where 690 00:46:22,630 --> 00:46:28,660 it gets chopped up by the serine protease active sites here. 691 00:46:28,660 --> 00:46:32,990 So for unfolding and translocation, ATP is needed. 692 00:46:32,990 --> 00:46:37,420 ClpX is hydrolyzing ATP to allow this to occur. 693 00:46:37,420 --> 00:46:39,970 In the degradation chamber, this degradation part 694 00:46:39,970 --> 00:46:41,270 is independent of ATP. 695 00:46:41,270 --> 00:46:41,770 Right? 696 00:46:41,770 --> 00:46:45,140 The serine protease doesn't need that here. 697 00:46:45,140 --> 00:46:47,830 So how can we kind of break this up further into a model 698 00:46:47,830 --> 00:46:49,600 that we can test? 699 00:46:49,600 --> 00:46:52,600 What I present here is the working model. 700 00:46:52,600 --> 00:46:56,320 And just note, the orientation is flipped here. 701 00:46:56,320 --> 00:47:00,910 So we have ClpX on the bottom and ClpP on top. 702 00:47:00,910 --> 00:47:04,270 So what happens here? 703 00:47:04,270 --> 00:47:08,380 We can look at this in terms of five steps. 704 00:47:08,380 --> 00:47:11,680 And we can begin here, with binding. 705 00:47:11,680 --> 00:47:16,150 So this ssrA-tagged protein needs to bind to ClpX. 706 00:47:16,150 --> 00:47:19,000 And that binding is associated with a dissociation 707 00:47:19,000 --> 00:47:23,050 constant, or Kd here. 708 00:47:23,050 --> 00:47:24,160 What do we see? 709 00:47:24,160 --> 00:47:28,930 After binding we have a second step, which is denaturation. 710 00:47:28,930 --> 00:47:33,550 So the polypeptide becomes unfolded. 711 00:47:33,550 --> 00:47:37,900 And that's defined by a rate constant for denaturation, 712 00:47:37,900 --> 00:47:40,220 as shown here. 713 00:47:40,220 --> 00:47:43,030 If we look next, we have translocation. 714 00:47:43,030 --> 00:47:46,840 So this polypeptide is moving through ClpX 715 00:47:46,840 --> 00:47:49,570 into the degradation chamber. 716 00:47:49,570 --> 00:47:53,230 And this is also associated with the rate constant-- 717 00:47:53,230 --> 00:47:55,450 so, rate constant for translocation. 718 00:47:55,450 --> 00:48:01,120 And both of these steps require the use of ATP. 719 00:48:01,120 --> 00:48:03,490 Once this polypeptide is in the chamber, 720 00:48:03,490 --> 00:48:07,410 we have step four, which is degradation. 721 00:48:07,410 --> 00:48:10,150 And again, we have k deg. 722 00:48:10,150 --> 00:48:11,440 This is fast. 723 00:48:11,440 --> 00:48:14,860 And then in this last step here, there's some release. 724 00:48:14,860 --> 00:48:16,900 So somehow these polypeptide fragments 725 00:48:16,900 --> 00:48:19,078 need to be released from the chamber. 726 00:48:19,078 --> 00:48:21,890 AUDIENCE: Is ClpP still a dimer at this point? 727 00:48:21,890 --> 00:48:22,880 PROFESSOR: Yes, yes. 728 00:48:22,880 --> 00:48:25,120 So often the cartoons are drawn just 729 00:48:25,120 --> 00:48:28,810 showing one of the heptamers. 730 00:48:28,810 --> 00:48:32,050 But think of it as a dimer, with these two back-to-back rings 731 00:48:32,050 --> 00:48:35,030 here for that. 732 00:48:35,030 --> 00:48:35,530 Right. 733 00:48:35,530 --> 00:48:38,830 So we have five steps here. 734 00:48:38,830 --> 00:48:41,650 Each one of these steps has a rate constant. 735 00:48:41,650 --> 00:48:45,220 And one question we want to ask with this is, what 736 00:48:45,220 --> 00:48:47,480 is the rate-determining step? 737 00:48:47,480 --> 00:48:50,680 And the quick answer where we'll end today, 738 00:48:50,680 --> 00:48:53,560 and as indicated in this overview, 739 00:48:53,560 --> 00:48:58,870 is that degradation is fast relative to denaturation 740 00:48:58,870 --> 00:49:00,990 and translocation. 741 00:49:00,990 --> 00:49:03,190 And there should be an intuitive aspect to that. 742 00:49:03,190 --> 00:49:05,530 We heard about last time how proteases 743 00:49:05,530 --> 00:49:08,230 give these tremendous rate accelerations. 744 00:49:08,230 --> 00:49:10,510 And if you have an unfolded peptide, 745 00:49:10,510 --> 00:49:12,370 those sites where cleavage will happen 746 00:49:12,370 --> 00:49:15,580 are going to be exposed there. 747 00:49:15,580 --> 00:49:17,860 So what we're going to ask is, is 748 00:49:17,860 --> 00:49:22,660 it possible to make experiments, design experience, where 749 00:49:22,660 --> 00:49:28,000 we can separate the denaturation process from the translocation 750 00:49:28,000 --> 00:49:30,970 process and analyze those-- 751 00:49:30,970 --> 00:49:33,910 and in the process of doing so, ask, 752 00:49:33,910 --> 00:49:36,440 what is the ATP utilization for each step? 753 00:49:36,440 --> 00:49:40,730 And what is the role for ATP in this process? 754 00:49:40,730 --> 00:49:44,130 And so on Friday we'll begin with discussing 755 00:49:44,130 --> 00:49:47,760 substrates, the design of substrates that have been used, 756 00:49:47,760 --> 00:49:50,340 to examine this model in more detail. 757 00:49:50,340 --> 00:49:51,800 Is there one question next? 758 00:49:51,800 --> 00:49:54,680 AUDIENCE: I was just wondering if the degradation step also 759 00:49:54,680 --> 00:49:58,880 removes translational modifications, or [INAUDIBLE] 760 00:49:58,880 --> 00:50:01,060 PROFESSOR: In the degradation step? 761 00:50:01,060 --> 00:50:02,460 That's going to depend. 762 00:50:02,460 --> 00:50:05,850 I mean, you can have different types of bonds 763 00:50:05,850 --> 00:50:08,650 with post-translational modifications, right? 764 00:50:08,650 --> 00:50:09,150 Right. 765 00:50:09,150 --> 00:50:10,950 So in the eukaryotic system, you have 766 00:50:10,950 --> 00:50:14,100 a post-translational modification 767 00:50:14,100 --> 00:50:17,070 to direct this condemned protein. 768 00:50:17,070 --> 00:50:19,650 And that machinery-- so they're ubiquitins, 769 00:50:19,650 --> 00:50:21,540 and you get this polyubiquitin tail. 770 00:50:21,540 --> 00:50:24,180 So you saw ubiquitin in recitation number one. 771 00:50:24,180 --> 00:50:26,400 And the eukaryotic proteasome has the ability 772 00:50:26,400 --> 00:50:29,250 to chop those ubiquitins ends off 773 00:50:29,250 --> 00:50:31,660 for recycling there, in that. 774 00:50:31,660 --> 00:50:34,070 So that's one example.