Effect of secondary interactions, steric hindrance and electric charge on the interaction of V(IV)O species with proteins

The interaction of two bis-chelated V(IV)O2+ species, formed by pipemidic (Hpip) and 8-hydroxyquinoline-5-sulphonic acids (H2hqs), with two small proteins, ubiquitin (Ub) and lysozyme (Lyz), was studied by EPR, ESI-MS, docking and DFT methods. The two complexes differ in electric charge (at neutral pH it is +2 and -2 for the species of Hpip and H2hqs, respectively), coordination geometry (cis-[VO(Hpip)2(H2O)]2+ is in equilibrium with the square pyramidal [VO(Hpip)2]2+, while cis-[VO(hqs)2(H2O)]2- is the only species in solution), and size (pipemidic acid is more sterically crowded than 8-hydroxyquinoline-5-sulphonic acid). The ESI-MS spectra showed that the number of cis-[VO(hqs)2]2- moieties which interact with Ub is twice that of cis-[VO(Hpip)2]2+ (adducts with formula n[VOL2]-Ub, where n = 1-4 for H2hqs and n = 1-2 for Hpip, were detected). The data can be rationalized in terms of the lower steric requirements of hqs2- and its negative charge, which favour secondary interactions with the surface groups of the protein. The EPR spectra revealed the coordination of Asp/Glu-COO or His-N donors, while docking calculations allowed us to identify the Ub residues involved in the VO(Hpip)2 (Asp39 and His68) and VO(hqs)2 binding (Glu18, Asp39, Asp58 and His68). The steric hindrance of Hpip is so important that the interaction of cis-[VO(Hpip)2(H2O)]2+ with lysozyme is completely prevented, while with cis-[VO(hqs)2(H2O)]2- the adducts n[VO(hqs)2]-Lyz (n = 1-2) were detected. Finally, the general effect of secondary interactions such as hydrogen bonds and van der Waals contacts, steric hindrance and electric charge on the V binding and stability of the adducts formed is also discussed, suggesting that these factors should be taken into account when designing new pharmacologically active compounds.