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![Page 1: Lab Meeting September 2001 John Wrobel. Outline Tour of HIV-1 RT DNA polymerization reaction pol “THE MOVIE” Role of AA residues in HIV-1 RT database.](https://reader035.fdocuments.in/reader035/viewer/2022062721/56649f1b5503460f94c31761/html5/thumbnails/1.jpg)
Lab MeetingSeptember 2001
John Wrobel
![Page 2: Lab Meeting September 2001 John Wrobel. Outline Tour of HIV-1 RT DNA polymerization reaction pol “THE MOVIE” Role of AA residues in HIV-1 RT database.](https://reader035.fdocuments.in/reader035/viewer/2022062721/56649f1b5503460f94c31761/html5/thumbnails/2.jpg)
Outline
• Tour of HIV-1 RT
• DNA polymerization reaction
• pol “THE MOVIE”
• Role of AA residues in HIV-1 RT database
![Page 3: Lab Meeting September 2001 John Wrobel. Outline Tour of HIV-1 RT DNA polymerization reaction pol “THE MOVIE” Role of AA residues in HIV-1 RT database.](https://reader035.fdocuments.in/reader035/viewer/2022062721/56649f1b5503460f94c31761/html5/thumbnails/3.jpg)
HIV-1 Reverse Transcriptase
p51
p66
heterodimer
![Page 4: Lab Meeting September 2001 John Wrobel. Outline Tour of HIV-1 RT DNA polymerization reaction pol “THE MOVIE” Role of AA residues in HIV-1 RT database.](https://reader035.fdocuments.in/reader035/viewer/2022062721/56649f1b5503460f94c31761/html5/thumbnails/4.jpg)
HIV-1 RT with DNA template
p66
p51
![Page 5: Lab Meeting September 2001 John Wrobel. Outline Tour of HIV-1 RT DNA polymerization reaction pol “THE MOVIE” Role of AA residues in HIV-1 RT database.](https://reader035.fdocuments.in/reader035/viewer/2022062721/56649f1b5503460f94c31761/html5/thumbnails/5.jpg)
HIV-1 RT with DNA template
p66
p51
![Page 6: Lab Meeting September 2001 John Wrobel. Outline Tour of HIV-1 RT DNA polymerization reaction pol “THE MOVIE” Role of AA residues in HIV-1 RT database.](https://reader035.fdocuments.in/reader035/viewer/2022062721/56649f1b5503460f94c31761/html5/thumbnails/6.jpg)
66 kd subunit
fingers
palm
connection
RNaseH
thumb
![Page 7: Lab Meeting September 2001 John Wrobel. Outline Tour of HIV-1 RT DNA polymerization reaction pol “THE MOVIE” Role of AA residues in HIV-1 RT database.](https://reader035.fdocuments.in/reader035/viewer/2022062721/56649f1b5503460f94c31761/html5/thumbnails/7.jpg)
HIV-1 RT subunits(primary sequence)
p66 fingers fingers thumbpalm palm connection RNaseH
fingers fingers thumbpalm palm connectionp51
1 85 120 151 243 323 438 560
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Conserved Sequence Motifs
Figure from CSH Symposia on Quant. Biol., Vol 53, pp 495-504 (1993)
Based on Protein Engineering 3, 461-467 (1990)
![Page 9: Lab Meeting September 2001 John Wrobel. Outline Tour of HIV-1 RT DNA polymerization reaction pol “THE MOVIE” Role of AA residues in HIV-1 RT database.](https://reader035.fdocuments.in/reader035/viewer/2022062721/56649f1b5503460f94c31761/html5/thumbnails/9.jpg)
Catalytic Site (aspartic acid triad)
D110D185D186
![Page 10: Lab Meeting September 2001 John Wrobel. Outline Tour of HIV-1 RT DNA polymerization reaction pol “THE MOVIE” Role of AA residues in HIV-1 RT database.](https://reader035.fdocuments.in/reader035/viewer/2022062721/56649f1b5503460f94c31761/html5/thumbnails/10.jpg)
p66 with DNA template
![Page 11: Lab Meeting September 2001 John Wrobel. Outline Tour of HIV-1 RT DNA polymerization reaction pol “THE MOVIE” Role of AA residues in HIV-1 RT database.](https://reader035.fdocuments.in/reader035/viewer/2022062721/56649f1b5503460f94c31761/html5/thumbnails/11.jpg)
Active Site for Polymerization
D185
D110
D186
E
F
9 10 6
AsparticAcid Triad
![Page 12: Lab Meeting September 2001 John Wrobel. Outline Tour of HIV-1 RT DNA polymerization reaction pol “THE MOVIE” Role of AA residues in HIV-1 RT database.](https://reader035.fdocuments.in/reader035/viewer/2022062721/56649f1b5503460f94c31761/html5/thumbnails/12.jpg)
Fingers – Secondary structureElement Sheet Residues
4 S2 W71 – D76
4 – B F77
B R78 – T84
C –D L120 – D123
D F124 – Y127
7 S1 T128 – P133
7 – 8 S134 – T139
8 S1 P140 – Y146
8 – E N147 – P150
Element Sheet Residues
P1 – G18
1 P19 – Q23
1 - A W24 – L26
A T27 – E44
A – 2 G45 – K46
2 S1 I47 – G51
2 – 3 P52 – Y56
3 S2 N57 – I63
3 – 4 K64 – K70
2 -sheets: S1, S23 -helices: A, B, D3 projecting loops: 21-45, 58-77, 130-144
![Page 13: Lab Meeting September 2001 John Wrobel. Outline Tour of HIV-1 RT DNA polymerization reaction pol “THE MOVIE” Role of AA residues in HIV-1 RT database.](https://reader035.fdocuments.in/reader035/viewer/2022062721/56649f1b5503460f94c31761/html5/thumbnails/13.jpg)
-helices A, B, D
Rasmol
Fingers
B
A
D
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-sheet 1
Rasmol
Fingers
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-sheet 2
Rasmol
Fingers
![Page 16: Lab Meeting September 2001 John Wrobel. Outline Tour of HIV-1 RT DNA polymerization reaction pol “THE MOVIE” Role of AA residues in HIV-1 RT database.](https://reader035.fdocuments.in/reader035/viewer/2022062721/56649f1b5503460f94c31761/html5/thumbnails/16.jpg)
Fingers
Rasmol
S2S1
A
D
B
![Page 17: Lab Meeting September 2001 John Wrobel. Outline Tour of HIV-1 RT DNA polymerization reaction pol “THE MOVIE” Role of AA residues in HIV-1 RT database.](https://reader035.fdocuments.in/reader035/viewer/2022062721/56649f1b5503460f94c31761/html5/thumbnails/17.jpg)
NRTI residues in 3-4
4
3
Rasmol
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NRTI residues in 3-4
4
3
Rasmol
K65D67T69K70L74V75
![Page 19: Lab Meeting September 2001 John Wrobel. Outline Tour of HIV-1 RT DNA polymerization reaction pol “THE MOVIE” Role of AA residues in HIV-1 RT database.](https://reader035.fdocuments.in/reader035/viewer/2022062721/56649f1b5503460f94c31761/html5/thumbnails/19.jpg)
2 loops involved in function
3-4
2-3(p66)
(p51)
![Page 20: Lab Meeting September 2001 John Wrobel. Outline Tour of HIV-1 RT DNA polymerization reaction pol “THE MOVIE” Role of AA residues in HIV-1 RT database.](https://reader035.fdocuments.in/reader035/viewer/2022062721/56649f1b5503460f94c31761/html5/thumbnails/20.jpg)
Rotation of Fingers
Structure 7, R31-R35 (1999)
3-4 loop bends 20°
Thick line = unliganded(open conformation)
Thin line = complexed with DNA(closed conformation)
![Page 21: Lab Meeting September 2001 John Wrobel. Outline Tour of HIV-1 RT DNA polymerization reaction pol “THE MOVIE” Role of AA residues in HIV-1 RT database.](https://reader035.fdocuments.in/reader035/viewer/2022062721/56649f1b5503460f94c31761/html5/thumbnails/21.jpg)
Region critical for protein stability in fingers subdomain of HIV-1 RT
p51
p66
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Region critical for protein stability in fingers subdomain of HIV-1 RT
78
loop
![Page 23: Lab Meeting September 2001 John Wrobel. Outline Tour of HIV-1 RT DNA polymerization reaction pol “THE MOVIE” Role of AA residues in HIV-1 RT database.](https://reader035.fdocuments.in/reader035/viewer/2022062721/56649f1b5503460f94c31761/html5/thumbnails/23.jpg)
Region critical for protein stability in fingers subdomain of HIV-1 RT
78
3
2
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Critical protein stability residue R143
7
8
R143
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Critical protein stability residue R143
R143
![Page 26: Lab Meeting September 2001 John Wrobel. Outline Tour of HIV-1 RT DNA polymerization reaction pol “THE MOVIE” Role of AA residues in HIV-1 RT database.](https://reader035.fdocuments.in/reader035/viewer/2022062721/56649f1b5503460f94c31761/html5/thumbnails/26.jpg)
Hydophilic Interactions
R143
N57
T131
Kinemage
![Page 27: Lab Meeting September 2001 John Wrobel. Outline Tour of HIV-1 RT DNA polymerization reaction pol “THE MOVIE” Role of AA residues in HIV-1 RT database.](https://reader035.fdocuments.in/reader035/viewer/2022062721/56649f1b5503460f94c31761/html5/thumbnails/27.jpg)
Hydrophobic residues critical for protein stability
I132
Y144
Y146
F130
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Big Picture
Kinemage
![Page 29: Lab Meeting September 2001 John Wrobel. Outline Tour of HIV-1 RT DNA polymerization reaction pol “THE MOVIE” Role of AA residues in HIV-1 RT database.](https://reader035.fdocuments.in/reader035/viewer/2022062721/56649f1b5503460f94c31761/html5/thumbnails/29.jpg)
Region critical for protein stability in fingers subdomain of HIV-1 RT
p66
p51
![Page 30: Lab Meeting September 2001 John Wrobel. Outline Tour of HIV-1 RT DNA polymerization reaction pol “THE MOVIE” Role of AA residues in HIV-1 RT database.](https://reader035.fdocuments.in/reader035/viewer/2022062721/56649f1b5503460f94c31761/html5/thumbnails/30.jpg)
Fingers
Residues 1-84
Residues 120-150
Rasmol
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Fig. 6-37 Voet
Hypothetical Folding Pathway
Denatured (unfolded protein)
Folding Intermediates Native (folded state)
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Fingers
Residues 1-84
Residues 120-150
Rasmol
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Palm – Secondary structureElement Sheet Residues
10 S3 D186 – S191
10 – F D192 – E194
F I195 – W212
F – 11 G213
11 S3 L214 – D218
11 – 12 K219 – P225
12 S4 P226 – M230
12 – 13 G231
13 S4 Y232 – H235
13 – 14 P236 – D237
14 S4 K238 – Q242
14 - H P243
Element Sheet Residues
B Q85
B – 5 D86 – L92
5 G93 – P97
5 – 6 A98 – K103
6 S3 K104 – G112
C D113 – V118
C – D P119
8 – E Q151 – W153
E K154 – Q174
E – 9 N175 – D177
9 S3 I178 – Y183
9 – 10 M184 – D185
2 -sheets: S3, S43 -helices: C, E, F
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-helices C, E, F
Rasmol
Palm
E
C
F
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-sheet 3
RasmolPalm
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Rasmol
-sheet 4
Palm
![Page 37: Lab Meeting September 2001 John Wrobel. Outline Tour of HIV-1 RT DNA polymerization reaction pol “THE MOVIE” Role of AA residues in HIV-1 RT database.](https://reader035.fdocuments.in/reader035/viewer/2022062721/56649f1b5503460f94c31761/html5/thumbnails/37.jpg)
Palm
Rasmol
S3
S4
C
F
E
![Page 38: Lab Meeting September 2001 John Wrobel. Outline Tour of HIV-1 RT DNA polymerization reaction pol “THE MOVIE” Role of AA residues in HIV-1 RT database.](https://reader035.fdocuments.in/reader035/viewer/2022062721/56649f1b5503460f94c31761/html5/thumbnails/38.jpg)
S191/H198 interaction
Kinemage
![Page 39: Lab Meeting September 2001 John Wrobel. Outline Tour of HIV-1 RT DNA polymerization reaction pol “THE MOVIE” Role of AA residues in HIV-1 RT database.](https://reader035.fdocuments.in/reader035/viewer/2022062721/56649f1b5503460f94c31761/html5/thumbnails/39.jpg)
Template Grip
Residue Region Subdomain
D76 4 Fingers
E89 B-5 Palm
Q151 8-E Palm
G152 8-E Palm
K154 8-E Palm
P157 E Palm
![Page 40: Lab Meeting September 2001 John Wrobel. Outline Tour of HIV-1 RT DNA polymerization reaction pol “THE MOVIE” Role of AA residues in HIV-1 RT database.](https://reader035.fdocuments.in/reader035/viewer/2022062721/56649f1b5503460f94c31761/html5/thumbnails/40.jpg)
Template Grip
KinemageBiopolymer 44, 125-138 (1997)
8-E loop: • Q151 & G152 interact with sugar-phosphate backbone of Tem-1 & Tem1• Main-chain atoms K154 with sugar-phosphate backbone of Tem1 & Tem2• P157 maintain b8-aE loop and position Q151, G152, K154
B –5 loop:• E89oe2 H-bonds with O3´ of Tem2
![Page 41: Lab Meeting September 2001 John Wrobel. Outline Tour of HIV-1 RT DNA polymerization reaction pol “THE MOVIE” Role of AA residues in HIV-1 RT database.](https://reader035.fdocuments.in/reader035/viewer/2022062721/56649f1b5503460f94c31761/html5/thumbnails/41.jpg)
Primer Grip
Residue Region Subdomain
W229 12-13 Palm
M230 12-13 Palm
G231 12-13 Palm
Y232 12-13 Palm
Kinemage
M230 & G231 interact with nucleotides of 3´-primer terminus
![Page 42: Lab Meeting September 2001 John Wrobel. Outline Tour of HIV-1 RT DNA polymerization reaction pol “THE MOVIE” Role of AA residues in HIV-1 RT database.](https://reader035.fdocuments.in/reader035/viewer/2022062721/56649f1b5503460f94c31761/html5/thumbnails/42.jpg)
dNTP Pocket
Structure = 1rtdScience 282, 1669-1675 (1998) Kinemage
• Triphosphate moiety is coordinated by K65, R72, main-chain –NH groups of D113 & A114• Guanidinium group of R72 lies flat against dNTP base & H-bonds with -phosphate• E-amino group of K65 H-bonds with g-phosphate• Main-chain –NH of Y115 H-bonds with O3* of dTTP
![Page 43: Lab Meeting September 2001 John Wrobel. Outline Tour of HIV-1 RT DNA polymerization reaction pol “THE MOVIE” Role of AA residues in HIV-1 RT database.](https://reader035.fdocuments.in/reader035/viewer/2022062721/56649f1b5503460f94c31761/html5/thumbnails/43.jpg)
Palm
Residues 85-119
Residues 151-243
Rasmol
![Page 44: Lab Meeting September 2001 John Wrobel. Outline Tour of HIV-1 RT DNA polymerization reaction pol “THE MOVIE” Role of AA residues in HIV-1 RT database.](https://reader035.fdocuments.in/reader035/viewer/2022062721/56649f1b5503460f94c31761/html5/thumbnails/44.jpg)
Thumb – Secondary structure
Element Sheet Residues
14 - H I244 – W252
H T253 – S268
H – I Q269 – K275
I V276 – K281
I – J L282 – E297
J E298 – L310
J – 15 K311 – V314
15 S4 H315 – Y319
15 – 16 D320 – D322
1 -sheet: S43 -helices: H, I, J
![Page 45: Lab Meeting September 2001 John Wrobel. Outline Tour of HIV-1 RT DNA polymerization reaction pol “THE MOVIE” Role of AA residues in HIV-1 RT database.](https://reader035.fdocuments.in/reader035/viewer/2022062721/56649f1b5503460f94c31761/html5/thumbnails/45.jpg)
-helices H, I, J
Rasmol
Thumb
H
I
J
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-sheet 4
Rasmol
Palm
Thumb
![Page 47: Lab Meeting September 2001 John Wrobel. Outline Tour of HIV-1 RT DNA polymerization reaction pol “THE MOVIE” Role of AA residues in HIV-1 RT database.](https://reader035.fdocuments.in/reader035/viewer/2022062721/56649f1b5503460f94c31761/html5/thumbnails/47.jpg)
Thumb
Rasmol
S4 H
I
J
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Primer-Template interactions with Thumb
KinemageBiopolymer 44, 125-138 (1997)
Helix H• Q258, K259, G262, K263, W266 vdw with sugar-phosphate backbone of Pri3 – Pri6• Q258ne2 H-bond with sugar O4´ atom of Pri6• K263nz salt bridge with phosphate O2P of Pri3• N265nd2 H-bond with ribose O3´ of Tem6
Helix I• S280, R284, G285, T286 vdw with sugar-phosphate backbone of Tem7 – Tem9 • Amide N of G285 H-bonds O1P & O2P of Tem9
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Unliganded RT (1dlo) – thumb folded into DNA-binding cleft
DNA-bound RT (2hmi) – thumb adopts an upright position
Flexibility of Thumb
Thumb’s knuckle = near residues W239 (14) & V317 (15)
Kinemage
Kinemage
![Page 50: Lab Meeting September 2001 John Wrobel. Outline Tour of HIV-1 RT DNA polymerization reaction pol “THE MOVIE” Role of AA residues in HIV-1 RT database.](https://reader035.fdocuments.in/reader035/viewer/2022062721/56649f1b5503460f94c31761/html5/thumbnails/50.jpg)
Connection – Secondary structureElement Sheet Residues
15 – 16 K323 – L325
16 S5 I326 – K331
16 – 17 Q332 – G335
17 S5 Q336 – Y342
17 – 18 Q343 – N348
18 S5 L349 – A355
18 – K R356 – N363
K D364 – W383
1 -sheet: S5 + S5A2 -helices: K, L
Element Sheet Residues
K – 19 G384 – T386
19 S5 P387 – L391
19 – L P392 – Q394
L K395 – E404
L – 20 Y405 – Q407
20 S5A A408 – P412
21 S5 E413 – N418
21 – R1 T419 – A437
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-helices K and L
Rasmol Connection
L
K
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-sheet 5
Rasmol
Connection
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Connection
Rasmol
S5 S5a
L
K
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Tryptophans in Connection
Rasmol
S5
S5a
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p66
p51
Dimer Interface
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Tryptophans at Dimer Interface
p66
p51Kinemage
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RNase H – Secondary structureElement Sheet Residues
R1 R1 E438 – N447
R1 – R2 R448 – K451
R2 R1 L452 – T459
R2 – R3 N460 – R461
R3 R1 G462 – T470
R3 – RA D471 – T473
RA N474 – D488
RA – RA S489 – L491
R4 R1 E492 – T497
1 -sheet: R14 -helices: aRA, RB, RD, RE
Element Sheet Residues
R4 - RB D498 – S499
RB Q500 – A508
RB - RD Q509 – S515
RD E516 – K527
RD – R5 K528 – E529
R5 R1 K530 – V536
R5 - RE P537 – G543
RE G544 – G555
I556 – L560
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-helices RA, RB, RD, RE
Rasmol
RBRNase H
RD
RE
RA
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-sheet R1
Rasmol
RNase H
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RNase H
Rasmol
SR1
RB
RD
RA
RE
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RNase H active site
H539(R5-RE)
D549(RE)
D443(R1)
E478(RA)
D498(R4-RB)
Rasmol
Kinemage
Kinemage
with DNA:
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-Helices in HIV-1 RTHelix Subdomain
A fingers
B fingers
C palm
D fingers
E palm
F palm
H thumb
I thumb
Helix Subdomain
J thumb
K connection
L connection
RA RNase H
RB RNase H
RD RNase H
RE RNase H
Total = 15 -helices
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-Sheets in HIV-1 RTSheet Strands Subdomain
S1 2, 7, 8 fingers
S2 3, 4 fingers
S3 6, 9, 10, 11 palm
S4 12, 13, 14, 15 palm/thumb
S5 16, 17, 18, 19, 21 connection
S5A 20 connection
R1 R1, R2, R3, R4, R5 RNase H
Total = 6 -sheets
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Action of DNA Polymerases
Voet Fig. 24-2
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Steps in DNA polymerization
• Binding of template-primer
• Binding of incoming dNTP
• Phosphodiester bond formation
• Release of pyrophosphate
• Translocation / Dissociation
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E E´—DNAn
Step 1 in DNA polymerization
Template-Primer binds to unliganded enzyme
DNAn
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E´—DNAn E´—DNAn—dNTP
Step 2 in DNA polymerization
Initiation of nucleotide incorporation
dNTP
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E´—DNAn—dNTP E*—DNAn—dNTP
Step 3 in DNA polymerization
Conversion to an activated complex
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E*—DNAn—dNTP E—DNAn+1
Step 4 in DNA polymerization
SN2 nucleophilic attack by the 3'-OH primer terminuson the -phosphate of dNTP resulting in phosphodiesterformation and removal of pyrophosphate product
PPi
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Nucleophilic attack by the 3' –OH catalyzes the phospho-Diester bond formation
Note that PPi is released
Action of DNA Polymerases- Another look
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Nucleotides
Science 264, 1891-1903 (1994)
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DNA Polymerization
Science 264, 1891-1903 (1994)
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Active Site for Polymerization
D185
D110
D186
E
F
9 10 6
AsparticAcid Triad
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HIV-1 RT: Polymerase Active Site
Arnold
Current Opinion in Structural Biology 5, 27-38 (1995)
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DNA polymerization at HIV-1 RT active site
Figure from CSH Symposia on Quant. Biol., Vol 53, pp 495-504 (1993)
Based on Protein Engineering 3, 461-467 (1990)
Steitz
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Model of DNA polymerization at HIV-1 RT active site
Journal of Biomolecular Structure & Dynamics12, 037-060 (1994)
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Model of HIV-1 RT polymerase active site
Journal of Biomolecular Structure & Dynamics12, 037-060 (1994)
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pol “THE MOVIE”
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Coming to a URL near you
http://chem-faculty.ucsd.edu/kraut/bpol.html
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Based on the Novel:
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pol
Smallest eukaryotic cellular DNA polymerase (39 kD)
Role: Fills single nucleotide gaps in DNA produced by the base excision pathway
pol has 2 subunits:• Nucleotidyl transfer activity (C-terminal 31 kD domain)
• Deoxyribosephosphate lyase activity (N-terminal 8 kD domain)
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Conformational changes of the THUMB during the catalytic cycle
Biochemistry 36, 11205-11215 (1997)
Watch for motion of Thumb & 8 kD domain
Gray = ternary complexBlack = binary complex
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Catalytic Aspartate 192
• With Thumb closure, F272 moves to disrupt R258-D192 H-bond• D192 binds Mg• E295 & Y296 position to H-bond with R258 (preventing R258 interference with D192)
Biochemistry 36, 11205-11215 (1997)
Gray = ternary complexBlack = binary complex
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dNTP position
With Thumb closure, H-bond donors of helix K (S180, R183, G189) interact with - and -phosphates of incoming dNTP
Biochemistry 36, 11205-11215 (1997)
Gray = ternary complexBlack = binary complex
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Template Position
Gray = ternary complexBlack = binary complex
Biochemistry 36, 11205-11215 (1997)
With Thumb closure, template is positioned to base-pair with dNTP
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Role of AA in HIV-1 RT DatabaseList of fields:
• Amino Acid: P1, I2, S3, P4, …. D110 … S191 … W401 … L560• Location: -helices, -sheets, loops, random coils• Sheet: -sheets• Subdomain: fingers, palm, thumb, connection, RNase H• Region: described in literature (example: primer-grip)• Motif: motif A, motif C• Role: from journal articles• Structure: role from structure papers• FSE: functional, stability, external residues (defined by HutchLab)• Eickbush alignment• Mutations: from other labs• HutchLab: mutations made by Hutchison lab• Inhibitor class: NNRTI, NRTI• Resistance
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John’s RT databases
• HIV-1 RT mutant data (phenotype & genotype) from HutchLab & others
• Role of Amino Acid Residues in the HIV-1 RT• HIV-1 RT H-bonds• HIV-1 RT van der Waals interactions• HIV-1 RT inhibitors• Retro RT H-bonds (from models, except MMLV)• Retro RT database (Eickbush alignment, variability)• Procam Results for HIV-1 and other retro RTs
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Alternative classificationscheme for the amino acids