Protein-ligand binding volume determined byFPSA, densitometry, and NMRZigmantas Toleikis§, Vladimir A. Sirotkin†, Gediminas Skvarnavicius§, Joana Smirnoviene§,Christian Roumestand‡, Daumantas Matulis§, and Vytautas Petrauskas§**E-mail: [email protected]§Department of Biothermodynamics and Drug Design, Institute of Biotechnology, Vilnius University, Vilnius, Lithuania†A.M. Butlerov Institute of Chemistry, Kazan Federal University, Kazan, Russia‡Centre de Biochimie Structurale, Université Montpellier I et II, Montpellier, France
XIV International Conference of the Lithuanian Biochemical Society | June 28 – 30, 2016 | Druskininkai, Lithuania
INTRODUCTIONWe report the values of recombinant human heat shock pro-tein 90 (Hsp90) binding volumes (i.e., the changes in proteinvolume associated with ligand binding), which were obtainedby three independent experimental techniques – fluorescentpressure shift assay (FPSA), vibrating tube densitometry, andhigh-pressure NMR. Within the error range all techniques pro-vide similar volumetric parameters of investigated protein-ligand systems.
LIGANDS
NH2 N SNH
N
N
N
Cl
N
OH
Cl
OOH
OH OH
O
O
OH
ICPD91 ICPD9 ICPD1
Cl
O
OO
OH
OH
O
OO
OH
OH
Cl
O
OO
OH
AZ1 AZ2 AZ3
OH
Cl
OH
N N
S
O
OH
OH
N N
S
O
OH
Cl
OH
O
OO
O
ICPD47 ICPD62 Radicicol
FLUORESCENT PRESSURE SHIFT ASSAY (FPSA)System of equations describing a protein dosing curve – the relationship between concentrationof added ligand, Lt, total protein concentration, Pt, and melting pressure, pm:
Lt = (exp(−∆GU/RT )− 1)
(Pt
2 exp(−∆GU/RT )+
1
exp(−∆Gb/RT )
), (1)
∆Gx = ∆G0_x + ∆Vx(pm − p0) +∆βx
2(pm − p0)
2; x = U, b, (2)
where ∆G0, ∆V and ∆β are standard state Gibbs energy, volume and compressibility factor,respectively, and indexes U and b stand for the changes related to protein unfolding and protein-ligand binding.
Flu
ore
scen
ce y
ield
(a.u
.)
1000
2000
3000
4000
p (MPa)0 100 200 300
Lt (μM of AZ2) 0 10 30 50
(a)
Δp m
(MP
a)
0
50
100
150
200
Lt (M)
0 10−6 10−5 10−4
Hsp90αN + ICPD1 AZ2 ICPD91 ICPD9
pur
e H
sp90
αN
(b)
(a)
FIGURE: (a) Unfolding profiles and (b) dosing curves of Hsp90αN.
HIGH-PRESSURE NMRThe Kd’s of Hsp90αN interaction with a ligand is calculatedfrom the chemical shift change, ∆δ, dependency on Lt and Pt:
∆δ = ∆δmaxY −
√Y 2 − 4LtPt
2Pt,
Y = Lt + Pt +Kd.
FIGURE: (a) ∆δ of Hsp90αN amino acids which were inducedby AZ3 and ICPD9 ligands. (b) ∆δ of Val92 as a function ofICPD9 concentration at 30 MPa, 120 MPa, and 150 MPa pres-sures. (c) AZ3-induced shifts in Hsp90αN (PDB ID: 1UYL).
DENSITOMETRY
The partial molar volume of a protein, V 0, and the change in protein volume associated with theligand binding, ∆Vb, are calculated using equations:
V 0 =M
d0− d− d0
Cd0, (3) V 0(R) = V 0(0) + α∆Vb, (4)
where M and C are molecular mass and molar concentration of a protein, d0 and d are densitiesof solvent and protein solution, respectively, and α is the fraction of ligand-bound protein:
α = 0.5 (1 +R+Kd/Pt)−√
0.25 (1 +R+Kd/)2 −R. (5)
Here Kd is the dissociation constant of the protein-ligand complex and R = Lt/Pt.
Solu
tio
n d
ensi
ty (g
/cm
3 )
0.9990
0.9992
0.9994
0.9996
0.9998
Ratio Lt / Pt
0 1 2 3 4
Hsp90αN + radicicol ICPD47 ICPD62 AZ3
(a)
V 0
(cm
3 /m
ol)
21360
21400
21440
21480
Ratio Lt / Pt
0 1 2 3 4
Hsp90αN + AZ3 ICPD47 ICPD62 radicicol
(b)
FIGURE: (a) Raw densitometry data and (b) the partial molar volumes of Hsp90αN at variousR.
BINDING VOLUMES
∆Vb (FPSA) ∆Vb (Densitometry) ∆Vb (NMR) Kd
ICPD91 −1± 5 cm3/mol n.d. n.d. 3× 10−5 MICPD9 −2± 5 cm3/mol n.d. 20± 4 cm3/mol 2× 10−5 MAZ3 −7± 6 cm3/mol −10± 3 cm3/mol −9± 4 cm3/mol 9× 10−5 MAZ2 −9± 6 cm3/mol n.d. n.d. 1× 10−3 MICPD1 −9± 6 cm3/mol n.d. n.d. 3× 10−5 MAZ1 −21± 11 cm3/mol n.d. n.d. 2× 10−3 MICPD47 −40± 14 cm3/mol −49± 5 cm3/mol n.d. 5× 10−9 MICPD62 n.d. −50± 4 cm3/mol n.d. 2× 10−9 Mradicicol −170± 60 cm3/mol −124± 7 cm3/mol n.d. 2× 10−10 M
ACKNOWLEDGEMENTThis research was funded by a grant (no. MIP-004/2014) fromthe Research Council of Lithuania.
