Lab%20Meeting%20September%202001

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Lab MeetingSeptember 2001

• John Wrobel

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Outline

• Tour of HIV-1 RT
• DNA polymerization reaction
• b pol THE MOVIE
• Role of AA residues in HIV-1 RT database

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HIV-1 Reverse Transcriptase
p66
heterodimer
p51
4
HIV-1 RT with DNA template
p66
p51
5
HIV-1 RT with DNA template
p66
p51
6
66 kd subunit
thumb
fingers
RNaseH
connection
palm
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HIV-1 RT subunits(primary sequence)
1
85
323
438
120
151
243
560
fingers
fingers
thumb
palm
palm
connection
RNaseH
p66
fingers
fingers
thumb
palm
palm
connection
p51
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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)
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Catalytic Site (aspartic acid triad)
D110 D185 D186
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p66 with DNA template
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Active Site for Polymerization
Aspartic Acid Triad
D186
D185
aE
D110
aF
b9
b10
b6
12
Fingers Secondary structure
Element Sheet Residues
b4 S2 W71 D76
b4 aB F77
aB R78 T84
aC aD L120 D123
aD F124 Y127
b7 S1 T128 P133
b7 b8 S134 T139
b8 S1 P140 Y146
b8 aE N147 P150
Element Sheet Residues
P1 G18
b1 P19 Q23
b1 - aA W24 L26
aA T27 E44
aA b2 G45 K46
b2 S1 I47 G51
b2 b3 P52 Y56
b3 S2 N57 I63
b3 b4 K64 K70
2 b-sheets S1, S2 3 a-helices aA, aB, aD 3
projecting loops 21-45, 58-77, 130-144
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a-helices A, B, D
Fingers
aA
aD
aB
Rasmol
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b-sheet 1
Fingers
Rasmol
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b-sheet 2
Fingers
Rasmol
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Fingers
aA
S2
S1
aB
aD
Rasmol
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NRTI residues in b3-b4
b3
b4
Rasmol
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NRTI residues in b3-b4
b3
b4
K65 D67 T69 K70 L74 V75
Rasmol
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2 loops involved in function
b3-b4
(p66)
b2-b3
(p51)
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Rotation of Fingers
b3-b4 loop bends 20
Thick line unliganded (open conformation) Thin
line complexed with DNA (closed conformation)
Structure 7, R31-R35 (1999)
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Region critical for protein stability in fingers
subdomain of HIV-1 RT
p66
p51
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Region critical for protein stability in fingers
subdomain of HIV-1 RT
loop
b7
b8
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Region critical for protein stability in fingers
subdomain of HIV-1 RT
b7
b8
b3
b2
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Critical protein stability residue R143
b7
R143
b8
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Critical protein stability residue R143
R143
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Hydophilic Interactions
T131
R143
Kinemage
N57
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Hydrophobic residues critical for protein
stability
I132
Y144
F130
Y146
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Big Picture
Kinemage
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Region critical for protein stability in fingers
subdomain of HIV-1 RT
p66
p51
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Fingers
Residues 1-84 Residues 120-150
Rasmol
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Hypothetical Folding Pathway
Folding Intermediates
Denatured (unfolded protein)
Native (folded state)
Fig. 6-37 Voet
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Fingers
Residues 1-84 Residues 120-150
Rasmol
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Palm Secondary structure
Element Sheet Residues
b10 S3 D186 S191
b10 aF D192 E194
aF I195 W212
aF b11 G213
b11 S3 L214 D218
b11 b12 K219 P225
b12 S4 P226 M230
b12 b13 G231
b13 S4 Y232 H235
b13 b14 P236 D237
b14 S4 K238 Q242
b14 - aH P243
Element Sheet Residues
aB Q85
aB b5 D86 L92
b5 G93 P97
b5 b6 A98 K103
b6 S3 K104 G112
aC D113 V118
aC aD P119
b8 aE Q151 W153
aE K154 Q174
aE b9 N175 D177
b9 S3 I178 Y183
b9 b10 M184 D185
2 b-sheets S3, S4 3 a-helices aC, aE, aF
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a-helices C, E, F
aE
aC
Palm
Rasmol
aF
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b-sheet 3
Palm
Rasmol
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b-sheet 4
Palm
Rasmol
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Palm
aE
aC
S3
aF
S4
Rasmol
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S191/H198 interaction
Kinemage
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Template Grip
Residue Region Subdomain
D76 b4 Fingers
E89 aB-b5 Palm
Q151 b8-aE Palm
G152 b8-aE Palm
K154 b8-aE Palm
P157 aE Palm
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Template Grip

• b8-aE 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

• aB b5 loop
• E89oe2 H-bonds with O3 of Tem2

Kinemage
Biopolymer 44, 125-138 (1997)
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Primer Grip
Residue Region Subdomain
W229 b12-b13 Palm
M230 b12-b13 Palm
G231 b12-b13 Palm
Y232 b12-b13 Palm
M230 G231 interact with nucleotides of
3-primer terminus
Kinemage
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dNTP Pocket

• 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 a-phosphate
• E-amino group of K65 H-bonds with g-phosphate
• Main-chain NH of Y115 H-bonds with O3 of dTTP

Structure 1rtd
Kinemage
Science 282, 1669-1675 (1998)
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Palm
Residues 85-119 Residues 151-243
Rasmol
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Thumb Secondary structure
Element Sheet Residues
b14 - aH I244 W252
aH T253 S268
aH aI Q269 K275
aI V276 K281
aI aJ L282 E297
aJ E298 L310
aJ b15 K311 V314
b15 S4 H315 Y319
b15 b16 D320 D322
1 b-sheet S4 3 a-helices aH, aI, aJ
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a-helices H, I, J
Thumb
aH
aJ
Rasmol
aI
46
b-sheet 4
Palm
Thumb
Rasmol
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Thumb
aH
S4
aJ
Rasmol
aI
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Primer-Template interactions with Thumb

• 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

Kinemage
Biopolymer 44, 125-138 (1997)
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Flexibility of Thumb
Kinemage
Unliganded RT (1dlo) thumb folded into
DNA-binding cleft DNA-bound RT (2hmi) thumb
adopts an upright position
Kinemage
Thumbs knuckle near residues W239 (b14) V317
(b15)
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Connection Secondary structure
Element Sheet Residues
b15 b16 K323 L325
b16 S5 I326 K331
b16 b17 Q332 G335
b17 S5 Q336 Y342
b17 b18 Q343 N348
b18 S5 L349 A355
b18 aK R356 N363
aK D364 W383
Element Sheet Residues
aK b19 G384 T386
b19 S5 P387 L391
b19 aL P392 Q394
aL K395 E404
aL b20 Y405 Q407
b20 S5A A408 P412
b21 S5 E413 N418
b21 bR1 T419 A437
1 b-sheet S5 S5A 2 a-helices aK, aL
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a-helices K and L
aL
aK
Rasmol
Connection
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b-sheet 5
Connection
Rasmol
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Connection
aK
aL
S5a
S5
Rasmol
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Tryptophans in Connection
S5
Rasmol
S5a
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Dimer Interface
p66
p51
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Tryptophans at Dimer Interface
p66
p51
Kinemage
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RNase H Secondary structure
Element Sheet Residues
bR1 R1 E438 N447
bR1 bR2 R448 K451
bR2 R1 L452 T459
bR2 bR3 N460 R461
bR3 R1 G462 T470
bR3 aRA D471 T473
aRA N474 D488
aRA bRA S489 L491
bR4 R1 E492 T497
Element Sheet Residues
bR4 - aRB D498 S499
aRB Q500 A508
aRB - aRD Q509 S515
aRD E516 K527
aRD bR5 K528 E529
bR5 R1 K530 V536
bR5 - aRE P537 G543
aRE G544 G555
I556 L560
1 b-sheet R1 4 a-helices aRA, aRB, aRD, aRE
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a-helices RA, RB, RD, RE
aRB
RNase H
aRE
aRD
Rasmol
aRA
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b-sheet R1
RNase H
Rasmol
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RNase H
aRD
aRB
aRA
aRE
SR1
Rasmol
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RNase H active site
D549 (aRE)
H539 (bR5-aRE)
D443 (bR1)
D498 (bR4-aRB)
E478 (aRA)
Kinemage
Rasmol
Kinemage
with DNA
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a-Helices in HIV-1 RT
Helix 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 a-helices
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b-Sheets in HIV-1 RT
Sheet Strands Subdomain
S1 b2, b7, b8 fingers
S2 b3, b4 fingers
S3 b6, b9, b10, b11 palm
S4 b12, b13, b14, b15 palm/thumb
S5 b16, b17, b18, b19, b21 connection
S5A b20 connection
R1 bR1, bR2, bR3, bR4, bR5 RNase H
Total 6 b-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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Step 1 in DNA polymerization
DNAn
E EDNAn
Template-Primer binds to unliganded enzyme
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Step 2 in DNA polymerization
dNTP
EDNAn EDNAndNTP
Initiation of nucleotide incorporation
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Step 3 in DNA polymerization
EDNAndNTP EDNAndNTP
Conversion to an activated complex
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Step 4 in DNA polymerization
PPi
EDNAndNTP EDNAn1
SN2 nucleophilic attack by the 3'-OH primer
terminus on the a-phosphate of dNTP resulting in
phosphodiester formation and removal of
pyrophosphate product
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Action of DNA Polymerases- Another look
Nucleophilic attack by the 3' OH catalyzes the
phospho- Diester bond formation Note that PPi
is released
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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
Aspartic Acid Triad
D186
D185
aE
D110
aF
b9
b10
b6
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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
Steitz
Figure from CSH Symposia on Quant. Biol., Vol 53,
pp 495-504 (1993) Based on Protein Engineering
3, 461-467 (1990)
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Model of DNA polymerization at HIV-1 RT active
site
Journal of Biomolecular Structure Dynamics 12,
037-060 (1994)
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Model of HIV-1 RT polymerase active site
Journal of Biomolecular Structure Dynamics 12,
037-060 (1994)
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pol b 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 b
Smallest eukaryotic cellular DNA polymerase (39
kD)

• pol b has 2 subunits
• Nucleotidyl transfer activity (C-terminal 31 kD
  domain)
• Deoxyribosephosphate lyase activity (N-terminal
  8 kD domain)

Role Fills single nucleotide gaps in DNA
produced by the base excision pathway
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Conformational changes of the THUMB during the
catalytic cycle
Gray ternary complex Black binary complex
Watch for motion of Thumb 8 kD domain
Biochemistry 36, 11205-11215 (1997)
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Movie 1
View
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Catalytic Aspartate 192
Gray ternary complex Black binary complex

• 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)
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Movie 2
View
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dNTP position
Gray ternary complex Black binary complex
With Thumb closure, H-bond donors of helix K
(S180, R183, G189) interact with b- and
g-phosphates of incoming dNTP
Biochemistry 36, 11205-11215 (1997)
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Movie 3
View
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Template Position
Gray ternary complex Black binary complex
With Thumb closure, template is positioned to
base-pair with dNTP
Biochemistry 36, 11205-11215 (1997)
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Movie 4
View
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Role of AA in HIV-1 RT Database

• List of fields
• Amino Acid P1, I2, S3, P4, . D110 S191
  W401 L560
• Location a-helices, b-sheets, loops, random
  coils
• Sheet b-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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Johns 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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(No Transcript)
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Alternative classificationscheme for the amino
acids