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Lab Meeting September 2001 John Wrobel Outline • Tour of HIV-1 RT • DNA polymerization reaction • b pol “THE MOVIE” • Role of AA residues in HIV-1 RT database HIV-1 Reverse Transcriptase p66 p51 heterodimer HIV-1 RT with DNA template p66 p51 HIV-1 RT with DNA template p66 p51 66 kd subunit thumb fingers RNaseH connection palm HIV-1 RT subunits (primary sequence) 1 85 120 151 323 243 438 p66 fingers palm fingers palm thumb connection p51 fingers palm fingers palm thumb connection 560 RNaseH 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) D110 D185 D186 p66 with DNA template Active Site for Polymerization D185 D186 aE D110 aF b9 b10 b6 Aspartic Acid Triad Fingers – Secondary structure Element Sheet Residues Element Sheet Residues P1 – G18 b4 b1 P19 – Q23 b4 – aB F77 b1 - aA W24 – L26 aB R78 – T84 aA T27 – E44 aC –aD L120 – D123 aA – b2 G45 – K46 aD F124 – Y127 I47 – G51 b7 P52 – Y56 b7 – b8 N57 – I63 b8 K64 – K70 b8 – aE b2 S1 b2 – b3 b3 b3 – b4 S2 S2 S1 W71 – D76 T128 – P133 S134 – T139 S1 P140 – Y146 N147 – P150 2 b-sheets: S1, S2 3 a-helices: aA, aB, aD 3 projecting loops: 21-45, 58-77, 130-144 a-helices A, B, D aA Fingers aD aB Rasmol b-sheet 1 Fingers Rasmol b-sheet 2 Fingers Rasmol Fingers aA S2 S1 aB Rasmol aD NRTI residues in b3-b4 b3 b4 Rasmol NRTI residues in b3-b4 b3 b4 Rasmol K65 D67 T69 K70 L74 V75 2 loops involved in function b3-b4 (p66) b2-b3 (p51) Rotation of Fingers b3-b4 loop bends 20° Thick line = unliganded (open conformation) Structure 7, R31-R35 (1999) Thin line = complexed with DNA (closed conformation) Region critical for protein stability in fingers subdomain of HIV-1 RT p66 p51 Region critical for protein stability in fingers subdomain of HIV-1 RT loop b8 b7 Region critical for protein stability in fingers subdomain of HIV-1 RT b7 b8 b3 b2 Critical protein stability residue R143 b7 R143 b8 Critical protein stability residue R143 R143 Hydophilic Interactions T131 R143 Kinemage N57 Hydrophobic residues critical for protein stability I132 F130 Y144 Y146 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 Hypothetical Folding Pathway Denatured (unfolded protein) Folding Intermediates Fig. 6-37 Voet Native (folded state) Fingers Residues 1-84 Residues 120-150 Rasmol Palm – Secondary structure Element Sheet Residues Element Sheet Residues aB Q85 b10 aB – b5 D86 – L92 b10 – aF D192 – E194 b5 G93 – P97 aF I195 – W212 b5 – b6 A98 – K103 aF – b11 G213 K104 – G112 b11 aC D113 – V118 b11 – b12 aC – aD P119 b12 b8 – aE Q151 – W153 b12 – b13 aE K154 – Q174 b13 aE – b9 N175 – D177 b13 – b14 I178 – Y183 b14 M184 – D185 b14 - aH b6 b9 b9 – b10 S3 S3 2 b-sheets: S3, S4 3 a-helices: aC, aE, aF S3 S3 D186 – S191 L214 – D218 K219 – P225 S4 P226 – M230 G231 S4 Y232 – H235 P236 – D237 S4 K238 – Q242 P243 a-helices C, E, F aE aC Palm Rasmol aF b-sheet 3 Palm Rasmol b-sheet 4 Palm Rasmol Palm aE aC S3 S4 Rasmol aF S191/H198 interaction Kinemage 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 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 Biopolymer 44, 125-138 (1997) Kinemage 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 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 Science 282, 1669-1675 (1998) Kinemage Palm Residues 85-119 Residues 151-243 Rasmol 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 b15 – b16 1 b-sheet: S4 3 a-helices: aH, aI, aJ H315 – Y319 D320 – D322 a-helices H, I, J Thumb aH aJ Rasmol aI b-sheet 4 Palm Thumb Rasmol Thumb S4 aH aJ Rasmol aI 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 Biopolymer 44, 125-138 (1997) Kinemage Flexibility of Thumb Kinemage Unliganded RT (1dlo) – thumb folded into DNA-binding cleft DNA-bound RT (2hmi) Kinemage – thumb adopts an upright position Thumb’s knuckle = near residues W239 (b14) & V317 (b15) Connection – Secondary structure Element Sheet Residues b15 – b16 Element Sheet Residues K323 – L325 aK – b19 I326 – K331 b19 Q332 – G335 b19 – aL P392 – Q394 Q336 – Y342 aL K395 – E404 Q343 – N348 aL – b20 Y405 – Q407 L349 – A355 b20 S5A A408 – P412 b18 – aK R356 – N363 b21 S5 E413 – N418 aK D364 – W383 b21 – bR1 b16 S5 b16 – b17 b17 S5 b17 – b18 b18 S5 1 b-sheet: S5 + S5A 2 a-helices: aK, aL G384 – T386 S5 P387 – L391 T419 – A437 a-helices K and L aL aK Rasmol Connection b-sheet 5 Connection Rasmol Connection aK aL S5 Rasmol S5a Tryptophans in Connection S5 Rasmol S5a Dimer Interface p66 p51 Tryptophans at Dimer Interface p66 p51 Kinemage RNase H – Secondary structure Element Sheet Residues bR1 R1 bR1 – bR2 bR2 R1 bR2 – bR3 bR3 R1 bR4 - aRB D498 – S499 R448 – K451 aRB Q500 – A508 L452 – T459 aRB - aRD Q509 – S515 N460 – R461 aRD E516 – K527 G462 – T470 aRD – bR5 K528 – E529 D471 – T473 aRA N474 – D488 aRA – bRA S489 – L491 R1 Sheet Residues E438 – N447 bR3 – aRA bR4 Element E492 – T497 bR5 R1 K530 – V536 bR5 - aRE P537 – G543 aRE G544 – G555 I556 – L560 1 b-sheet: R1 4 a-helices: aRA, aRB, aRD, aRE a-helices RA, RB, RD, RE aRB RNase H aRE Rasmol aRA aRD b-sheet R1 RNase H Rasmol RNase H aRD aRA aRB aRE SR1 Rasmol RNase H active site D549 (aRE) D443 H539 (bR5-aRE) D498 (bR1) (bR4-aRB) E478 (aRA) Kinemage Rasmol with DNA: Kinemage 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 RNase H RA RNase H RB RNase H RD RNase H RE Total = 15 a-helices b-Sheets in HIV-1 RT Sheet Strands S1 S2 S3 S4 S5 S5A R1 Subdomain b2, b7, b8 fingers b3, b4 fingers b6, b9, b10, b11 palm b12, b13, b14, b15 palm/thumb b16, b17, b18, b19, b21 connection b20 connection bR1, bR2, bR3, bR4, bR5 RNase H Total = 6 b-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 Step 1 in DNA polymerization DNAn E E´—DNAn Template-Primer binds to unliganded enzyme Step 2 in DNA polymerization dNTP E´—DNAn E´—DNAn—dNTP Initiation of nucleotide incorporation Step 3 in DNA polymerization E´—DNAn—dNTP E*—DNAn—dNTP Conversion to an activated complex Step 4 in DNA polymerization PPi E*—DNAn—dNTP E—DNAn+1 SN2 nucleophilic attack by the 3'-OH primer terminus on the a-phosphate of dNTP resulting in phosphodiester formation and removal of pyrophosphate product Action of DNA Polymerases- Another look Nucleophilic attack by the 3' –OH catalyzes the phosphoDiester bond formation Note that PPi is released Nucleotides Science 264, 1891-1903 (1994) DNA Polymerization Science 264, 1891-1903 (1994) Active Site for Polymerization D185 D186 aE D110 aF b9 b10 b6 Aspartic Acid 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 Steitz Figure from CSH Symposia on Quant. Biol., Vol 53, pp 495-504 (1993) Based on Protein Engineering 3, 461-467 (1990) Model of DNA polymerization at HIV-1 RT active site Journal of Biomolecular Structure & Dynamics 12, 037-060 (1994) Model of HIV-1 RT polymerase active site Journal of Biomolecular Structure & Dynamics 12, 037-060 (1994) pol b “THE MOVIE” Coming to a URL near you http://chem-faculty.ucsd.edu/kraut/bpol.html Based on the Novel: 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 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) Movie 1 View 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) Movie 2 View 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) Movie 3 View Template Position Gray = ternary complex Black = binary complex With Thumb closure, template is positioned to base-pair with dNTP Biochemistry 36, 11205-11215 (1997) Movie 4 View 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 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 classification scheme for the amino acids
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