lab meeting september 2001 john wrobel. outline tour of hiv-1 rt dna polymerization reaction pol...
TRANSCRIPT
Lab MeetingSeptember 2001
John Wrobel
Outline
• Tour of HIV-1 RT
• DNA polymerization reaction
• pol “THE MOVIE”
• Role of AA residues in HIV-1 RT database
HIV-1 Reverse Transcriptase
p51
p66
heterodimer
HIV-1 RT with DNA template
p66
p51
HIV-1 RT with DNA template
p66
p51
66 kd subunit
fingers
palm
connection
RNaseH
thumb
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
Conserved Sequence Motifs
Figure from CSH Symposia on Quant. Biol., Vol 53, pp 495-504 (1993)
Based on Protein Engineering 3, 461-467 (1990)
Catalytic Site (aspartic acid triad)
D110D185D186
p66 with DNA template
Active Site for Polymerization
D185
D110
D186
E
F
9 10 6
AsparticAcid Triad
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
-helices A, B, D
Rasmol
Fingers
B
A
D
-sheet 1
Rasmol
Fingers
-sheet 2
Rasmol
Fingers
Fingers
Rasmol
S2S1
A
D
B
NRTI residues in 3-4
4
3
Rasmol
NRTI residues in 3-4
4
3
Rasmol
K65D67T69K70L74V75
2 loops involved in function
3-4
2-3(p66)
(p51)
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)
Region critical for protein stability in fingers subdomain of HIV-1 RT
p51
p66
Region critical for protein stability in fingers subdomain of HIV-1 RT
78
loop
Region critical for protein stability in fingers subdomain of HIV-1 RT
78
3
2
Critical protein stability residue R143
7
8
R143
Critical protein stability residue R143
R143
Hydophilic Interactions
R143
N57
T131
Kinemage
Hydrophobic residues critical for protein stability
I132
Y144
Y146
F130
Big Picture
Kinemage
Region critical for protein stability in fingers subdomain of HIV-1 RT
p66
p51
Fingers
Residues 1-84
Residues 120-150
Rasmol
Fig. 6-37 Voet
Hypothetical Folding Pathway
Denatured (unfolded protein)
Folding Intermediates Native (folded state)
Fingers
Residues 1-84
Residues 120-150
Rasmol
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
-helices C, E, F
Rasmol
Palm
E
C
F
-sheet 3
RasmolPalm
Rasmol
-sheet 4
Palm
Palm
Rasmol
S3
S4
C
F
E
S191/H198 interaction
Kinemage
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
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
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
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
Palm
Residues 85-119
Residues 151-243
Rasmol
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
-helices H, I, J
Rasmol
Thumb
H
I
J
-sheet 4
Rasmol
Palm
Thumb
Thumb
Rasmol
S4 H
I
J
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
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
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
-helices K and L
Rasmol Connection
L
K
-sheet 5
Rasmol
Connection
Connection
Rasmol
S5 S5a
L
K
Tryptophans in Connection
Rasmol
S5
S5a
p66
p51
Dimer Interface
Tryptophans at Dimer Interface
p66
p51Kinemage
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
-helices RA, RB, RD, RE
Rasmol
RBRNase H
RD
RE
RA
-sheet R1
Rasmol
RNase H
RNase H
Rasmol
SR1
RB
RD
RA
RE
RNase H active site
H539(R5-RE)
D549(RE)
D443(R1)
E478(RA)
D498(R4-RB)
Rasmol
Kinemage
Kinemage
with DNA:
-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
-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
Action of DNA Polymerases
Voet Fig. 24-2
Steps in DNA polymerization
• Binding of template-primer
• Binding of incoming dNTP
• Phosphodiester bond formation
• Release of pyrophosphate
• Translocation / Dissociation
E E´—DNAn
Step 1 in DNA polymerization
Template-Primer binds to unliganded enzyme
DNAn
E´—DNAn E´—DNAn—dNTP
Step 2 in DNA polymerization
Initiation of nucleotide incorporation
dNTP
E´—DNAn—dNTP E*—DNAn—dNTP
Step 3 in DNA polymerization
Conversion to an activated complex
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
Nucleophilic attack by the 3' –OH catalyzes the phospho-Diester bond formation
Note that PPi is released
Action of DNA Polymerases- Another look
Nucleotides
Science 264, 1891-1903 (1994)
DNA Polymerization
Science 264, 1891-1903 (1994)
Active Site for Polymerization
D185
D110
D186
E
F
9 10 6
AsparticAcid Triad
HIV-1 RT: Polymerase Active Site
Arnold
Current Opinion in Structural Biology 5, 27-38 (1995)
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
Model of DNA polymerization at HIV-1 RT active site
Journal of Biomolecular Structure & Dynamics12, 037-060 (1994)
Model of HIV-1 RT polymerase active site
Journal of Biomolecular Structure & Dynamics12, 037-060 (1994)
pol “THE MOVIE”
Coming to a URL near you
http://chem-faculty.ucsd.edu/kraut/bpol.html
Based on the Novel:
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)
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
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
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
Template Position
Gray = ternary complexBlack = binary complex
Biochemistry 36, 11205-11215 (1997)
With Thumb closure, template is positioned to base-pair with dNTP
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
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
Alternative classificationscheme for the amino acids