Anti-HDX monoclonal antibody Made to order

The hydrogen deuterium exchange (HDX) mass spectrometry combined with molecular dynamics (MD) simulations was employed to investigate conformational dynamics and ligand binding within the M49 family (dipeptidyl peptidase III family). Six dipeptidyl peptidase III (DPP III) orthologues, human, yeast, three bacterial and one plant (moss) were studied. According to the results, all orthologues seem to be quite compact wherein DPP III from the thermophile Caldithrix abyssi seems to be the most compact. The protected regions are located within the two domains core and the overall flexibility profile consistent with semi-closed conformation as the dominant protein form in solution. Besides conservation of conformational dynamics within the M49 family, we also investigated the ligand, pentapeptide tynorphin, binding. By comparing HDX data obtained for unliganded protein with those obtained for its complex with tynorphin it was found that the ligand binding mode is conserved within the family. Tynorphin binds within inter-domain cleft, close to the lower domain -core and induces its stabilization in all orthologues. Docking combined with MD simulations revealed details of the protein flexibility as well as of the enzyme-ligand interactions.

Allosteric HIV-1 integrase (IN) inhibitors (ALLINIs) bind at the dimer interface of the IN catalytic core domain (CCD), and potently inhibit HIV-1 by promoting aberrant, higher-order IN multimerization. Little is known about the structural organization of the inhibitor-induced IN multimers and important questions regarding how ALLINIs promote aberrant IN multimerization remain to be answered. On the basis of physical chemistry principles and from our analysis of experimental information, we propose that inhibitor-induced multimerization is mediated by ALLINIs directly promoting inter-subunit interactions between the CCD dimer and a C-terminal domain (CTD) of another IN dimer. Guided by this hypothesis, we have built atomic models of inter-subunit interfaces in IN multimers by incorporating information from hydrogen-deuterium exchange (HDX) measurements to drive protein-protein docking. We have also developed a novel free energy simulation method to estimate the effects of ALLINI binding on the association of the CCD and CTD. Using this structural and thermodynamic modeling approach, we show that multimer inter-subunit interface models can account for several experimental observations about ALLINI-induced multimerization, including large differences in the potencies of various ALLINIs, the mechanisms of resistance mutations, and the crucial role of solvent exposed R-groups in the high potency of certain ALLINIs. Our study predicts that CTD residues Tyr226, Trp235 and Lys266 are involved in the aberrant multimer interfaces. The key finding of the study is that it suggests the possibility of ALLINIs facilitating inter-subunit interactions between an external CTD and the CCD-CCD dimer interface.