PHEMTO: protein pH-dependent electric moment tools

Aykut

The Journal of Chemical Physics

2011

This study reveals the essence of ligand recognition mechanisms by which calmodulin (CaM) controls a variety of Ca(2+) signaling processes. We study eight forms of calcium-loaded CaM each with distinct conformational states. Reducing the structure to two degrees of freedom conveniently describes main features of the conformational changes of CaM via simultaneous twist-bend motions of the two lobes. We utilize perturbation-response scanning (PRS) technique, coupled with molecular dynamics simulations. PRS is based on linear response theory, comprising sequential application of directed forces on selected residues followed by recording the resulting protein coordinates. We analyze directional preferences of the perturbations and resulting conformational changes. Manipulation of a single residue reproduces the structural change more effectively than that of single/pairs/triplets of collective modes of motion. Our findings also give information on how the flexible linker acts as a transducer of binding information to distant parts of the protein. Furthermore, by perturbing residue E31 located in one of the EF hand motifs in a specific direction, it is possible to induce conformational change relevant to five target structures. Independently, using four different pK(a) calculation strategies, we find this particular residue to be the charged residue (out of a total of 52), whose ionization state is most sensitive to subtle pH variations in the physiological range. It is plausible that at relatively low pH, CaM structure is less flexible. By gaining charged states at specific sites at a pH value around 7, such as E31 found in the present study, local conformational changes in the protein will lead to shifts in the energy landscape, paving the way to other conformational states. These findings are in accordance with Fluorescence Resonance Energy Transfer (FRET) measured shifts in conformational distributions towards more compact forms with decreased pH. They also corroborate mutational studies and proteolysis results which point to the significant role of E31 in CaM dynamics.

Section: Resultsmentioning

confidence: 99%

Subtle pH differences trigger single residue motions for moderating conformations of calmodulin

Aykut

The Journal of Chemical Physics

2011

Journal of Thrombosis and Haemostasis

“…We also used the atomic coordinates of the C2 contained in the PDB file of the Fab BO2C11 ‐C2 complex, which we refer to as ‘separated’ C2 domain. Electrostatic calculation was performed using a home‐made software package PHEI and PHEMTO server [18–21], using the mean force approach, written in Perl and C/C++ with Haskell extensions. The program allows calculation of the pH dependence of: (i) global electrostatic characteristics of proteins or macromolecular complexes – electrostatic free energy ΔE el (pH), protein net charge DZ(pH), electrostatic potential Φ el , and value and direction of the dipole/electric moments vectors μ d /μ el (pH)); (ii) local electrostatic characteristics such as degree of ionization of all titrable groups S i (pH), and electrostatic energy of interaction of each ionic group with the whole charged multipol E el,i (pH); and (iii) proton affinity at each individual ionic site pK a,i (pH) and local electrostatic potential ϕ i (pH).…”

Section: Methodsmentioning

confidence: 99%

A human FVIII inhibitor modulates FVIII surface electrostatics at a VWF‐binding site distant from its epitope

Dimitrov

Roumenina

et al. 2010

Summary. Background: BO2C11 is a human monoclonal factor (F) VIII inhibitor. When bound to the C2 domain of FVIII, the Fab fragment of BO2C11 (Fab BO2C11 ) buries a surface of C2 that contains residues participating in a binding site for von Willebrand factor (VWF). BO2C11 has thus been proposed to neutralize FVIII by steric hindrance. Objectives: The BO2C11 epitope on C2 overlaps with residues located at the periphery of the putative VWF binding site; hence, most of the residues that constitute the VWF binding site on C2 and a3 remain accessible for VWF interaction following BO2C11/ FVIII complex formation. We thus investigated the contribution of alternative molecular mechanisms to FVIII inactivation by BO2C11. Methods: Continuum electrostatic calculations were applied to the crystal structure of C2, free or Fab BO2C11 -complexed. In silico predictions were confirmed by site-directed mutagenesis and VWF-binding assays of the mutated FVIII. Results: Binding of Fab BO2C11 to C2 induced perturbations in the electrostatic potential of C2 and in the local electrostatic parameters of 18 charged residues in C2, which are distant from the BO2C11 epitope. Nine of the predicted electrostatic hotspots clustered on the VWF-binding site of C2. Mutation of some of the predicted electrostatic hotspots has been associated with hemophilia A and reduced VWF binding in vitro. Conclusions: Inhibitors may neutralize FVIII by alteration of protein surface electrostatics at a long distance from their epitope. Perturbation of the electrostatic environment of C2, either upon binding by anti-FVIII antibodies or consecutive to missense mutations in the F8 gene, may lead to hampered VWF binding and reduced FVIII residence time in circulation.

Journal of Biological Chemistry

“…Electric moments determinations were performed using the PHEMTO sever (Institute of Organic Chemistry, Biophysical Chemistry Lab at Bulgarian Academy of Sciences) as described previously (34,35). PHEMTO (PH-dependent Electric Moments of proteins TOols) uses three different approaches to calculate three-dimensional electrostatic potentials grid(s) of proteins and their complexes.…”

Section: Electrostatic Analysis Of Gc1q-heme Complexmentioning

confidence: 99%

Heme Interacts with C1q and Inhibits the Classical Complement Pathway

Roumenina

Radanova

Атанасов

et al. 2011

Self Cite

C1q is the recognition subunit of the first component of the classical complement pathway. It participates in clearance of immune complexes and apoptotic cells as well as in defense against pathogens. Inappropriate activation of the complement contributes to cellular and tissue damage in different pathologies, urging the need for the development of therapeutic agents that are able to inhibit the complement system. In this study, we report heme as an inhibitor of C1q. Exposure of C1q to heme significantly reduced the activation of the classical complement pathway, mediated by C-reactive protein (CRP) and IgG. Interaction analyses revealed that heme reduces the binding of C1q to CRP and IgG. Furthermore, we demonstrated that the inhibition of C1q interactions results from a direct binding of heme to C1q. Formation of complex of heme with C1q caused changes in the mechanism of recognition of IgG and CRP. Taken together, our data suggest that heme is a natural negative regulator of the classical complement pathway at the level of C1q. Heme may play a role at sites of excessive tissue damage and hemolysis where large amounts of free heme are released.