A single-point mutation of M-PMV matrix protein causes reorientation of protein domains and changes the phenotype of the virus Jiří Vlach1, Jan Lipov2, Václav Veverka1, Jan Lang1,3, Pavel Srb1,3, Michaela Rumlová4, Eric Hunter5, Tomáš Ruml2,4 and Richard Hrabal1 1
Laboratory of NMR Spectroscopy 2Department of Biochemistry and Microbiology @Institute of Chemical Technology Prague, Prague, Czech Republic 3Department of Low Temperature Physics, Faculty of Mathematics and Physics, Charles University, Prague, Czech Republic 4Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Prague, Czech Republic 5Yerkes Natural Primate Research Center, Emory Vaccine Center, Atlanta GA USA
Životní cyklus retrovirů
➢ Comparison of B/D and C type retroviruses electron microscopy images of infected cells
B/D type (M-PMV)
C type (HIV-1)
Mature Virus Particle SU envelope CA NC
MA
TM
env gag
genome
Gag precursor MA
p24
p12
CA
NC
p4
➢ Structure determination sample
homogeneously 13C and 15N 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 13C/15N 3D NOESY
structure calculation
restraints: calculation:
NOE dihedral angles , (Talos), H-bonds (CSI) Aria 2.0alpha (NOE assignment, calibration) NIH-Xplor (simulated annealing)
➢ Structures of wild type MA and R55F mutant C
N
wild type matrix
N
C
RMSD = 0.48 Å
R55F mutant
N
N
C C RMSD = 0.59 Å
➢ Comparison of the CTRS regions wild type matrix
residue 55 = Arg
R55F mutant
residue 55 = Phe
➢ Cytoplasmic dynein minus end directed molecular motor use of ATP transport of celular cargos along microtubules
o c rg a
stalk
heavy chain (2x530 kDa)
head stem
intermediate chains (2x74, 4x55 kDa) light chains (ca. 10 kDa) several families, Tctex-1 proved to interact with wt MA
plus end
minus end
Oligomerization of Non-myristoylated M-PMV Matrix Protein J. Vlach1,2, P. Srb3,1, M. Grocký3, J. Lang3,1, J. Prchal1,2, E. Hunter4, T. Ruml2, R. Hrabal1
1Laboratory
of NMR Spectroscopy
2Department
of Biochemistry and Microbiology @ Institute of Chemical Technology, Prague
3Department
of Low Temperature Physics, Faculty of Mathematics and Physics, Charles University 4Emory
Vaccine Center, Yerkes National Primate Research Center
Oligomerization of matrix proteins crystal SIV, HIV-1
trimeric
EIAV, MoMuLV
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, 11364–11369
HIV-1 MA
←
Tang et al. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 517–522
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)
Results: chemical shift analysis concentration series of 8 MA samples 1 15N HSQC spectra ●measured H●
calculation of chemical shift changes: combined chemical shift differences, CCSD ●
𝐶𝐶𝑆𝐷 =
Δδ2H +
Δδ2N 25
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, 10565–10570
Results: chemical shift analysis Histogram of chemical shift changes (c0 vs. c0/40) Selected residues: T41 W44 F45 D61 C62 D65 Y66 Y67 T69 F70
Concentration dependence of CCSDs
Results: translational diffusion coefficients (D)
1H
processing in Gifa software
homonuclear method (double stimulated spin echo)
Concentration dependence of D (in H2O) HydroNMRcalculated D D1 = 105 D2 = 80 D3 = 70 (10–12 m2s–1)
Determination of the oligomerization model Concentration dependence of populations
approximately 55 % of MA is in an oligomeric state
Calculation of structures of oligomers HADDOCK (high ambiguity-driven biomolecular docking) input:
M-PMV dimer
concentration-affected residues symmetry restraints M-PMV trimer
HIV-1 trimer
Comparison of M-PMV and HIV-1 MA trimers
M-PMV MA trimer “top”
MA trimerization interfaces
HIV-1
ERFAVNQQQTGSEE
M-PMV
TWFDCDYYTF
“bottom”
orange = oligomerization capacity
Possible biological implications myristic acid
M-PMV procapsid
MA pp24 p12
MA
CA
CA
NC p4
NC
M-PMV Gag
HIV-1 Gag
(D-type)
(C-type)
M-PMV MA trimerization: stabilization of Gag N terminus in assembled procapsids
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 (13C/15N) NOESY experimenty
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
Mason-Pfizer monkey virus (M-PMV) Phospholipid membrane Matrix protein (MA) Capsid protein Nucleocapsid protein Genomic RNA
Transmembrane glycoprotein Surface glycoprotein
M-PMV matrix protein (MA) N-terminus
Mw 11969 Da
100 amino acids N-terminally myristoylated
C-terminus
Vlach J. et al. PNAS, 2008
HIV-1 MA interacting with membrane
Phosphatidylinositol(4,5) bisphosphate (PIP) Saad et al. 2006
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
Cleveage site for M-PMV protease
His-tag
Myristoylation induced large chemical shift changes 1H15N-HSQC
(backbone)
MAPPHi s myrMAPPH is
W56 I5 3
R5 7 Y6 7
X V10 3
1H-13C-HSQC
(side-chains) myrMAPPHis
MAPPHis
13C-filtered /13C-edited
NOESY
NOE kontakty CH3 skupiny kyseliny myristové chemický posun δ (CH3) = 0,8 ppm
I86 (CH3 δ)
CH3
I51 (CH3 δ)
<5Å <5Å I51
X
I51 (CH3 γ)
I86
1H
I86 (CH3 γ)
13C
Structure of myrMA
Comparison of myristoylated and non-myristoylated MA
N
N
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
Interaction of MA with PIP
1H-15N
and 1H-13C combined chemical shift changes of MA residues 13C-filtered 13C-edited
NOESY spectra
Chemical shift changes of phosphor from PIP
Saturation Transfer Difference (STD)
Titrace MyrMA PI(4,5)P2 sledovaná pomocí 1H-15N-HSQC
Titrace MyrMA PI(4,5)P2 sledovaná pomocí 1H-15N-HSQC
𝐶𝐶𝑆𝐷 =
Δδ2H +
Δδ2N 25
Interaction of myrMA with PIP Largest chemical – shift changes
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 KD
STD-experiments
STD – MA+PIP
PIP
31P
chemical shift changes
High Ambiguity Driven DOCKing HADDOCK
Complex of PIP with myrMA
Complex of PIP with myrMA
Interaction of MA with PIP 0,6
Δδ 0,5
myrMA – L31
0,4
0,3
0,2
MA – L31
0,1
0 0
0,5
1
1,5
2
2,5
3
3,5
4
4,5
c[PIP]/c[MA]
Myristoylated MA
non-myristoylated MA
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 (13C/15N) NOESY experimenty
Transferred nuclear Overhauser effect
E
+
L
EL
[ E ][ L] koff Kd [ EL] kon
Transferred nuclear Overhauser effect
H
H NOE
r<5Å
chemická výměna
H
r>5Å
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 (Mw > 40 kDa) neviditelný pro NMR
Podmínka: Využití:
vhodná kinetika systému 10-8 > Kd > 10-3 M-1 - struktura ligandu - nepřímo struktura vazebného místa - způsob vazby
Elegantní metoda pro design nových typů léčiv
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
TM52-1+5C
C-E-A-P-E-G-Y-I-L-D-D-G-F-I-C-T-D-I-D-E
TM52+2+5C
E-C-P-E-G-Y-I-G-D-x- x-F-x-C-T-D-I-D-E
TM52-3+6C
C-P-E-G-Y-F-G-D-D-G-S-x-C-T-D-I
TM52-1+4C
NMR vzorek: a) ligand, Mw ≈ 2 kDa, konc. 0.7 mM Bovine thrombin, Mw ≈ 40 kDa, konc. 0.07 mM, poměr k peptidu 1 : 10 b) ligand Mw ≈ 2 kDa, konc. 0.7 mM Bovine prothrombin (prekursor), Mw ≈ 70 kDa, konc. 0.0035 mM, poměr k peptidu 1 : 20 NMR experimenty: a) 1H –titrace peptidu thrombinem (prothrombinem) b) clean-TOCSY pro přiřazení rezonancí c) NOESY (WATERGATE)
Titrace ligandu roztokem thrombinu
Důvod: testování specifické vazby substrátu a ligandu
A B C
volný peptid TM52+5C TM52+5C + bovine thrombin (1:10) TM52+5C + bovine prothhrombin (1:20)
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
Důležité NOE interakce dalekého dosahu ligandu TM52+5C v komplexu s thrombinem
Srovnání experimentálního a vypočteného NOESY spektra jako kriterium kvality určené struktury
Experiment
Výpočet
Struktura komplexu thrombinu Complex between thrombin and TM52+5Ca ligandu TM52+5C
Ile
Arg
