Major research themes
recognition and cleavage mechanism of DNA
action of intrinsically unstructured proteins
We employ and develop computational techniques to investigate the mechanism of specific binding of proteins to DNA and elucidate the molecular choreography and catalytic factors of DNA-breaking reactions, which are central to all DNA processing biological events. Using bioinformatical approaches and simulation methodology we aim to uncover the origin of specific and efficient binding of intrinsically unstructured protein segments to their partners.
We investigate how proteins localize their cognate DNA sites/sequences focusing on those factors that govern the initial stages of the recognition process. Upon scanning for their target sites, proteins can decode sequential information of the DNA even in the absence of direct contacts with the base-pairs. We propose that conversion of the initial encounter complex to the specific complex is controlled by three elements: 1) structure of the interfacial water (hydration fingerprint), 2) local/global flexibility of the DNA, 3) bivalent metal ion binding. We analyze specific water structure around different DNA sequences/damages and identify first contact points for the partner protein based on local decrease in hydration, water binding energies and residence times. We also relate local water release from a given site to its contribution to the binding free energy of the complex. We investigate local flexibility changes around DNA lesions along specific coordinates that facilitate distorsions required for binding to their repair enzyme. We attempt to uncover how divalent metal cation localization on DNA such as Mg2+ and Mn2+ affect selectivity of restriction enzymes.
DNA cleavage mechanism
DNA-breaking reactions are central steps in the maintenance, repair and expression of genetic information. To understand the catalysis of this process, type II restriction enzymes are employed as model systems. Biochemical and structural data provide alternative catalytic mechanisms for the PD..D/ExK enzymes with similar active site and three dimensional structure. The PD..D/ExK architecture is not exquisite to restriction enzymes, but also characteristic to other nucleases involved in DNA repair. The main uncertainty is in the number of metal ions cofactors involved in the phosphodiester hydrolysis reaction and the identity of the general base that produces the attacking nucleophile. We aim to develop a uniform scenario for DNA-cleavage by PD..D/ExK restriction endonucleases via quantitative analysis of the main catalytic factors. To this end we apply hybrid quantum mechanical/molecular mechanical (QM/MM) techniques to elaborate the reaction mechanism and determine the contribution of different residues/metal ions to the activation free energy. We also probe the stability of metal ion positions by molecular dynamics (MD) simulations and free energy calculations.
Action of intrinsically unstructured proteins
Numerous experimental results evidence the functioning of proteins without a well-defined three dimensional structure, often via a binding-coupled folding process. In general, partner recognition of these intrinsically unstructured proteins (IUPs) or disordered regions (IDRs) can be characterized by high specificity, low affinity and favorable on and off kinetics. In collaboration with Peter Tompa, we aim to identify those molecular features that code the selectivity of IUPs/IDPs and enhance their binding efficiency. We investigate the role of pre-existing recognition motifs, such as pre-formed secondary structure elements and primary binding sites in the interaction of IUPs/IDPs with their partners. We also study how the action of short, linear motifs mediating protein-protein contacts is related to the malleability of their embedding environment.