1. Lab Meeting September 2001 John Wrobel

2. Outline Tour of HIV-1 RT DNA polymerization reaction  pol “THE MOVIE” Role of AA residues in HIV-1 RT database

3. HIV-1 Reverse Transcriptase p51 p66 heterodimer

4. HIV-1 RT with DNA template p66 p51

5. HIV-1 RT with DNA template p66 p51

6. 66 kd subunit fingers palm connection RNaseH thumb

7. HIV-1 RT subunits (primary sequence) p66 fingers thumbpalm connectionRNaseH fingers thumbpalm connection p51 185 120151243 323438 560

8. Conserved Sequence Motifs Figure from CSH Symposia on Quant. Biol., Vol 53, pp 495-504 (1993) Based on Protein Engineering 3, 461-467 (1990)

9. Catalytic Site (aspartic acid triad) D110 D185 D186

10. p66 with DNA template

11. Active Site for Polymerization D185 D110 D186 EE FF 99  10 66 Aspartic Acid Triad

12. Fingers – Secondary structure ElementSheetResidues 44 S2W71 – D76  4 –  B F77 BB R78 – T84  C –  D L120 – D123 DD F124 – Y127 77 S1T128 – P133  7 –  8 S134 – T139 88 S1P140 – Y146  8 –  E N147 – P150 ElementSheetResidues P1 – G18 11 P19 – Q23  1 -  A W24 – L26 AA T27 – E44  A –  2 G45 – K46 22 S1I47 – G51  2 –  3 P52 – Y56 33 S2N57 – I63  3 –  4 K64 – K70 2  -sheets: S1, S2 3  -helices:  A,  B,  D 3 projecting loops: 21-45, 58-77, 130-144

13.  -helices A, B, D Rasmol Fingers BB AA DD

14.  -sheet 1 Rasmol Fingers

15.  -sheet 2 Rasmol Fingers

16. Rasmol S2 S1 AA DD BB

17. NRTI residues in  3-  4 44 33 Rasmol

18. NRTI residues in  3-  4 44 33 Rasmol K65 D67 T69 K70 L74 V75

19. 2 loops involved in function  3-  4  2-  3 (p66) (p51)

20. 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)

21. Region critical for protein stability in fingers subdomain of HIV-1 RT p51 p66

22. Region critical for protein stability in fingers subdomain of HIV-1 RT 77 88 loop

23. Region critical for protein stability in fingers subdomain of HIV-1 RT 77 88 33 22

24. Critical protein stability residue R143 77 88 R143

25. Critical protein stability residue R143 R143

26. Hydophilic Interactions R143 N57 T131 Kinemage

27. Hydrophobic residues critical for protein stability I132 Y144 Y146 F130

28. Big Picture Kinemage

29. Region critical for protein stability in fingers subdomain of HIV-1 RT p66 p51

30. Fingers Residues 1-84 Residues 120-150 Rasmol

31. Fig. 6-37 Voet Hypothetical Folding Pathway Denatured (unfolded protein) Folding Intermediates Native (folded state)

32. Fingers Residues 1-84 Residues 120-150 Rasmol

33. Palm – Secondary structure ElementSheetResidues  10 S3D186 – S191  10 –  F D192 – E194 FF I195 – W212  F –  11 G213  11 S3L214 – D218  11 –  12 K219 – P225  12 S4P226 – M230  12 –  13 G231  13 S4Y232 – H235  13 –  14 P236 – D237  14 S4K238 – Q242  14 -  H P243 ElementSheetResidues BB Q85  B –  5 D86 – L92 55 G93 – P97  5 –  6 A98 – K103 66 S3K104 – G112 CC D113 – V118  C –  D P119  8 –  E Q151 – W153 EE K154 – Q174  E –  9 N175 – D177 99 S3I178 – Y183  9 –  10 M184 – D185 2  -sheets: S3, S4 3  -helices:  C,  E,  F

34.  -helices C, E, F Rasmol Palm EE CC FF

35.  -sheet 3 Rasmol Palm

36. Rasmol  -sheet 4 Palm

37. Rasmol S3 S4 CC FF EE

38. S191/H198 interaction Kinemage

39. Template Grip ResidueRegionSubdomain D76 44 Fingers E89  B-  5 Palm Q151  8-  E Palm G152  8-  E Palm K154  8-  E Palm P157 EE Palm

40. Template Grip Kinemage Biopolymer 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 O 3´ of Tem2

41. Primer Grip ResidueRegionSubdomain 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

42. dNTP Pocket Structure = 1rtd Science 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

43. Palm Residues 85-119 Residues 151-243 Rasmol

44. Thumb – Secondary structure ElementSheetResidues  14 -  H I244 – W252 HH T253 – S268  H –  I Q269 – K275 II V276 – K281  I –  J L282 – E297 JJ E298 – L310  J –  15 K311 – V314  15 S4H315 – Y319  15 –  16 D320 – D322 1  -sheet: S4 3  -helices:  H,  I,  J

45.  -helices H, I, J Rasmol Thumb HH II JJ

46.  -sheet 4 Rasmol Palm Thumb

47. Rasmol S4 HH II JJ

48. Primer-Template interactions with Thumb Kinemage Biopolymer 44, 125-138 (1997) Helix H Q258, K259, G262, K263, W266 vdw with sugar-phosphate backbone of Pri3 – Pri6 Q258ne2 H-bond with sugar O 4´ atom of Pri6 K263nz salt bridge with phosphate O 2P of Pri3 N265nd2 H-bond with ribose O 3´ of Tem6 Helix I S280, R284, G285, T286 vdw with sugar-phosphate backbone of Tem7 – Tem9 Amide N of G285 H-bonds O 1P & O 2P of Tem9

49. 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

50. Connection – Secondary structure ElementSheetResidues  15 –  16 K323 – L325  16 S5I326 – K331  16 –  17 Q332 – G335  17 S5Q336 – Y342  17 –  18 Q343 – N348  18 S5L349 – A355  18 –  K R356 – N363 KK D364 – W383 1  -sheet: S5 + S5A 2  -helices:  K,  L ElementSheetResidues  K –  19 G384 – T386  19 S5P387 – L391  19 –  L P392 – Q394 LL K395 – E404  L –  20 Y405 – Q407  20 S5AA408 – P412  21 S5E413 – N418  21 –  R1 T419 – A437

51.  -helices K and L Rasmol Connection LL KK

52.  -sheet 5 Rasmol Connection

53. Rasmol S5 S5a LL KK

54. Tryptophans in Connection Rasmol S5 S5a

55. p66 p51 Dimer Interface

56. Tryptophans at Dimer Interface p66 p51 Kinemage

57. RNase H – Secondary structure ElementSheetResidues R1R1 R1E438 – N447  R 1 –  R 2 R448 – K451 R2R2 R1L452 – T459  R 2 –  R 3 N460 – R461 R3R3 R1G462 – T470  R 3 –  R A D471 – T473 RARA N474 – D488  R A –  R A S489 – L491 R4R4 R1E492 – T497 1  -sheet: R1 4  -helices: a R A,  R B,  R D,  R E ElementSheetResidues  R 4 -  R B D498 – S499 RBRB Q500 – A508  R B -  R D Q509 – S515 RDRD E516 – K527  R D –  R 5 K528 – E529 R5R5 R1K530 – V536  R 5 -  R E P537 – G543 RERE G544 – G555 I556 – L560

58.  -helices R A, R B, R D, R E Rasmol RBRB RNase H RDRD RERE RARA

59.  -sheet R 1 Rasmol RNase H

60. Rasmol SR1SR1 RBRB RDRD RARA RERE

61. RNase H active site H539 (  R 5-  R E) D549 (  R E) D443 (  R 1) E478 (  R A) D498 (  R 4-  R B) Rasmol Kinemage with DNA:

62.  -Helices in HIV-1 RT HelixSubdomain Afingers B Cpalm Dfingers Epalm F Hthumb I HelixSubdomain Jthumb Kconnection L RARARNase H RBRB RDRD RERE Total = 15  -helices

63.  -Sheets in HIV-1 RT SheetStrandsSubdomain 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  R 1,  R 2,  R 3,  R 4,  R 5 RNase H Total = 6  -sheets

64. Action of DNA Polymerases Voet Fig. 24-2

65. Steps in DNA polymerization Binding of template-primer Binding of incoming dNTP Phosphodiester bond formation Release of pyrophosphate Translocation / Dissociation

66. E E´—DNA n Step 1 in DNA polymerization Template-Primer binds to unliganded enzyme DNA n

67. E´—DNA n E´—DNA n —dNTP Step 2 in DNA polymerization Initiation of nucleotide incorporation dNTP

68. E´—DNA n —dNTP E*—DNA n —dNTP Step 3 in DNA polymerization Conversion to an activated complex

69. E*—DNA n —dNTP E—DNA n+1 Step 4 in DNA polymerization SN2 nucleophilic attack by the 3'-OH primer terminus on the  -phosphate of dNTP resulting in phosphodiester formation and removal of pyrophosphate product PPi

70. Nucleophilic attack by the 3' –OH catalyzes the phospho- Diester bond formation Note that PPi is released Action of DNA Polymerases- Another look

71. Nucleotides Science 264, 1891-1903 (1994)

72. DNA Polymerization Science 264, 1891-1903 (1994)

73. Active Site for Polymerization D185 D110 D186 EE FF 99  10 66 Aspartic Acid Triad

74. HIV-1 RT: Polymerase Active Site Arnold Current Opinion in Structural Biology 5, 27-38 (1995)

75. 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

76. Model of DNA polymerization at HIV-1 RT active site Journal of Biomolecular Structure & Dynamics 12, 037-060 (1994)

77. Model of HIV-1 RT polymerase active site Journal of Biomolecular Structure & Dynamics 12, 037-060 (1994)

78. pol  “THE MOVIE”

79. Coming to a URL near you http://chem-faculty.ucsd.edu/kraut/bpol.html

80. Based on the Novel:

81. 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)

82. Conformational changes of the THUMB during the catalytic cycle Biochemistry 36, 11205-11215 (1997) Watch for motion of Thumb & 8 kD domain Gray = ternary complex Black = binary complex

83. Movie 1 View

84. 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 complex Black = binary complex

85. Movie 2 View

86. 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 complex Black = binary complex

87. Movie 3 View

88. Template Position Gray = ternary complex Black = binary complex Biochemistry 36, 11205-11215 (1997) With Thumb closure, template is positioned to base-pair with dNTP

89. Movie 4 View

90. Role of AA in HIV-1 RT Database List 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

91. 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

93. Alternative classification scheme for the amino acids