A single-point mutation of M-PMV matrix protein causes reorientation of protein domains and changes the phenotype of the virus

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1 A single-point mutation of M-PMV matrix protein causes reorientation of protein domains and changes the phenotype of the virus Jiří Vlach 1, Jan Lipov 2, Václav Veverka 1, Jan Lang 1,3, Pavel Srb 1,3, Michaela Rumlová 4, Eric Hunter 5, Tomáš Ruml 2,4 and Richard Hrabal 1 1 Laboratory of NMR Spectroscopy 2 Department of Biochemistry and of Chemical Technology Prague, Prague, Czech Republic 3 Department of Low Temperature Physics, Faculty of Mathematics and Physics, Charles University, Prague, Czech Republic 4 Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Prague, Czech Republic 5 Yerkes Natural Primate Research Center, Emory Vaccine Center, Atlanta GA USA

2 Životní cyklus retrovirů

3 Comparison of B/D and C type retroviruses electron microscopy images of infected cells B/D type (M-PMV) C type (HIV-1)

4 Mature Virus Particle SU envelope TM env CA NC MA gag genome Gag precursor MA p24 p12 CA NC p4

5 Structure determination sample homogeneously 13 C and 15 N enriched protein vector pet22b, E. coli strain BL21, growth in a minimal medium final concentration 1.0 mmol l 1 NMR spectroscopy Bruker DRX-500 Avance NMR spectrometer resonance assignment: HNCO, HNCA, HN(CO)CA, HNCACB, CBCA(CO)NH, HB(CB)HA(CA)(CO)NH; H(C)CH-TOCSY, (H)CCH-COSY NOE distance restraints: edited 13 C/ 15 N 3D NOESY structure calculation restraints: calculation: NOE dihedral angles, (Talos), H-bonds (CSI) Aria 2.0alpha (NOE assignment, calibration) NIH-Xplor (simulated annealing)

6 Structures of wild type MA and R55F mutant wild type matrix N C N C RMSD = 0.48 Å R55F mutant N N C C RMSD = 0.59 Å

7 Comparison of the CTRS regions wild type matrix R55F mutant residue 55 = Arg residue 55 = Phe

Cytoplasmic dynein minus end directed molecular motor use of ATP transport of celular cargos along microtubules stalk head stem heavy chain (2x530

8 Cytoplasmic dynein minus end directed molecular motor use of ATP transport of celular cargos along microtubules stalk head stem heavy chain (2x530 kda) ca rgo intermediate chains (2x74, 4x55 kda) light chains (ca. 10 kda) several families, Tctex-1 proved to interact with wt MA plus end minus end

10 Oligomerization of Non-myristoylated M-PMV Matrix Protein J. Vlach 1,2, P. Srb 3,1, M. Grocký 3, J. Lang 3,1, J. Prchal 1,2, E. Hunter 4, T. Ruml 2, R. Hrabal 1 1 Laboratory of NMR Spectroscopy 2 Department of Biochemistry and Institute of Chemical Technology, Prague 3 Department of Low Temperature Physics, Faculty of Mathematics and Physics, Charles University 4 Emory Vaccine Center, Yerkes National Primate Research Center

11 Oligomerization of matrix proteins crystal SIV, HIV-1 EIAV, MoMuLV trimeric non-trimeric solution HIV-1, EIAV monomer trimer HIV-1 myr( )-MA monomer HIV-2, RSV monomer M-PMV myr( )-MA? Saad et al. (2006) Proc. Natl. Acad. Sci. U. S. A. 103, HIV-1 MA Tang et al. (2004) Proc. Natl. Acad. Sci. U. S. A. 101,

12 Methods for study of oligomerization analytical ultracentrifugation dynamic light scattering calorimetry nuclear magnetic resonance (NMR) chemical shifts concentration dependence translational diffusion effective size rotational diffusion (NMR relaxation)

13 Results: chemical shift analysis concentration series of 8 MA samples measured 1 H- 15 N HSQC spectra calculation of chemical shift changes: combined chemical shift differences, CCSD CCSD = Δδ H 2 + Δδ N 2 25

14 Study of R55F MA mutant oligomerization WT MA R55F MA residue residue C C WT MA N R55F MA N Vlach et al. (2008) Proc. Natl. Acad. Sci. U. S. A. 105,

15 Results: chemical shift analysis Histogram of chemical shift changes (c 0 vs. c 0 /40) Selected residues: T41 W44 F45 D61 C62 D65 Y66 Y67 T69 F70 Concentration dependence of CCSDs

16 Results: translational diffusion coefficients (D) 1 H homonuclear method (double stimulated spin echo) processing in Gifa software Concentration dependence of D (in H 2 O) HydroNMRcalculated D D 1 = 105 D 2 = 80 D 3 = 70 (10 12 m 2 s 1 )

17 Determination of the oligomerization model Concentration dependence of populations approximately 55 % of MA is in an oligomeric state

18 Calculation of structures of oligomers HADDOCK (high ambiguity-driven biomolecular docking) input: concentration-affected residues symmetry restraints M-PMV dimer M-PMV trimer HIV-1 trimer

19 Comparison of M-PMV and HIV-1 MA trimers top M-PMV MA trimer MA trimerization interfaces HIV-1 M-PMV ERFAVNQQQTGSEE TWFDCDYYTF bottom

20 Possible biological implications orange = oligomerization capacity myristic acid MA M-PMV procapsid pp24 p12 CA MA CA NC p4 M-PMV Gag (D-type) NC HIV-1 Gag (C-type) M-PMV MA trimerization: stabilization of Gag N terminus in assembled procapsids

22 NMR spektroskopie komplexů Co lze studovat? Struktury dvou nebo více interagujících částí komplexu Vzájemná orientace interagujících částí Určení parametrů, které charakterizují komplex (disociační konstanta, rychlost přístupu, event. odstupu interagujících částí Metodické postupy: Izotopově neobohacené molekuly Transferred Nuclear Overhauser Experiment - TRNOE Izotopově obohacený ligand nebo receptor Izotopově filtrované ( 13 C/ 15 N) NOESY experimenty

23 VŠCHT PRAHA Structure of Myristoylated Matrix Protein of Mason-Pfizer Monkey Virus and Role of Phosphatidylinositol-(4,5)- bisphosphate in its Membrane Binding Jan Prchal, Tomas Ruml, Richard Hrabal

24 Mason-Pfizer monkey virus (M-PMV) Phospholipid membrane Matrix protein (MA) Capsid protein Nucleocapsid protein Genomic RNA Transmembrane glycoprotein Surface glycoprotein

25 M-PMV matrix protein (MA) N-terminus Mw Da 100 amino acids N-terminally myristoylated C-terminus Vlach J. et al. PNAS, 2008

26 HIV-1 MA interacting with membrane Phosphatidylinositol(4,5) bisphosphate (PIP) Saad et al. 2006

27 Studied protein MA with 20 AA from pp24 and His-tag on C-terminus Myristoylated MA is not cleaved by M-PMV Pr Model for MA in immature virus particle myr MA 18 AA of PP His-tag Cleveage site for M-PMV protease

28 Myristoylation induced large chemical shift changes 1 H 15 N-HSQC (backbone) MAPPHi s myrmapph is R5 7 W56 I5 3 Y6 7 X V10 3

29 1 H- 13 C-HSQC (side-chains) myrmapphis MAPPHis

30 13 C-filtered / 13 C-edited NOESY NOE kontakty CH 3 skupiny kyseliny myristové chemický posun δ (CH 3 ) = 0,8 ppm < 5 Å CH 3 I86 (CH 3 δ) I51 (CH 3 δ) I51 X < 5 Å I86 I51 (CH 3 γ) I86 (CH 3 γ) 13 C 1 H

31 Structure of myrma

32 Comparison of myristoylated and non-myristoylated MA N N

33 Phosphatidylinositolphosphates PI(3)P early endosomes PI(3,5)P late endozomes PI(4)P Golgi complex PI(4,5)P Cytoplasmic membrane PI(3,5)P, PI(3,4,5)P signal molecules

34 Interaction of MA with PIP 1 H- 15 N and 1 H- 13 C combined chemical shift changes of MA residues 13 C-filtered 13 C-edited NOESY spectra Chemical shift changes of phosphor from PIP Saturation Transfer Difference (STD)

35 Titrace MyrMA PI(4,5)P 2 sledovaná pomocí 1 H- 15 N-HSQC

36 Titrace MyrMA PI(4,5)P 2 sledovaná pomocí 1 H- 15 N-HSQC CCSD = Δδ H 2 + Δδ N 2 25

37 Interaction of myrma with PIP Largest chemical shift changes

38 STD Saturation transfer difference Interakce protein-malá molekula Ozáření proteinu a přenos magnetizace na ligand během interakce Přebytek ligandu Zjistíme, která část ligandu interaguje Odhad K D

39 STD-experiments STD MA+PIP PIP

40 31 P chemical shift changes

41 High Ambiguity Driven DOCKing HADDOCK

42 Complex of PIP with myrma

43 Complex of PIP with myrma

44 Interaction of MA with PIP Δδ 0,6 0,5 myrma L31 0,4 0,3 0,2 0,1 MA L ,5 1 1,5 2 2,5 3 3,5 4 4,5 c[pip]/c[ma]

45 Myristoylated MA non-myristoylated MA

47 NMR spektroskopie komplexů Co lze studovat? Struktury dvou nebo více interagujících částí komplexu Vzájemná orientace interagujících částí Určení parametrů, které charakterizují komplex (disociační konstanta, rychlost přístupu, event. odstupu interagujících částí Metodické postupy: Izotopově neobohacené molekuly Transferred Nuclear Overhauser Experiment - TRNOE Izotopově obohacený ligand nebo receptor Izotopově filtrované ( 13 C/ 15 N) NOESY experimenty

48 Transferred nuclear Overhauser effect E + L EL K d [ E][ L] [ EL] k k off on

49 Transferred nuclear Overhauser effect r < 5 Å H H NOE chemická výměna r > 5 Å H H Princip: informace o struktuře ligandu ve vázaném stavu je pomocí chemické výměny přenesena na ligand ve volném stavu, kde je detekována Uspořádání: malý ligand, který je viditelný NMR spektroskopií a velký substrát (M w > 40 kda) neviditelný pro NMR Podmínka: vhodná kinetika systému 10-8 > K d > 10-3 M -1 Využití: - struktura ligandu - nepřímo struktura vazebného místa - způsob vazby Elegantní metoda pro design nových typů léčiv

53 Uspořádání experimentu A. Peptidové fragmenty z thrombomodulinu (5. EGF) C-P-E-G-Y-I-L-D-D-G-F-I-C-T-D-I-D-E TM52+5C C-P-E-G-Y-I-L-D-D-G-F-x-C-T-D-I-D-E C-E-A-P-E-G-Y-I-L-D-D-G-F-I-C-T-D-I-D-E E-C-P-E-G-Y-I-G-D-x- x-f-x-c-t-d-i-d-e C-P-E-G-Y-F-G-D-D-G-S-x-C-T-D-I TM52-1+5C TM52+2+5C TM52-3+6C TM52-1+4C NMR vzorek: a) ligand, M w 2 kda, konc. 0.7 mm Bovine thrombin, M w 40 kda, konc mm, poměr k peptidu 1 : 10 b) ligand M w 2 kda, konc. 0.7 mm Bovine prothrombin (prekursor), M w 70 kda, konc mm, poměr k peptidu 1 : 20 NMR experimenty: a) 1 H titrace peptidu thrombinem (prothrombinem) b) clean-tocsy pro přiřazení rezonancí c) NOESY (WATERGATE)

54 Titrace ligandu roztokem thrombinu Důvod: testování specifické vazby substrátu a ligandu A volný peptid TM52+5C B TM52+5C + bovine thrombin (1:10) C TM52+5C + bovine prothhrombin (1:20)

55 NOESY spektrum peptidu TM52+5C ve volném a vázaném stavu. Možná struktura ligandu ve volném stavu nesmi interferovat se strukturou ve stavu vázaném!!! NOESY spektrum komplexu TM52+5C s thrombinem NOESY spektrum volného TM52+5C

56 Důležité NOE interakce dalekého dosahu ligandu TM52+5C v komplexu s thrombinem

58 Srovnání experimentálního a vypočteného NOESY spektra jako kriterium kvality určené struktury Experiment Výpočet

59 Struktura Complex komplexu between thrombin thrombinu and TM52+5C a ligandu TM52+5C Ile Arg

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