MocR bacterial transcriptional regulators (TRs) are a subfamily of the larger GntR family. The proteins of this family possess a characteristic molecular architecture: an N-terminal DNA-binding domain containing a winged Helix-Turn-Helix (wHTH) motif and a C-terminal domain with oligomerization and/or effector binding function, interconnected through a linker of variable length. The MocR subfamily is characterised by a C-terminal domain homologous to the fold type I pyridoxal 5¿-phosphate (PLP)-dependent enzymes (such as aspartate aminotransferase), connected to the wHTH domain by a linker of variable length. The PLP cofactor is covalently bound to an active site lysine residue via a Schiff base. The project aims at studying the functional and structural properties of the MocR-TRs using in-silico techniques and it is focused on one particular regulator, namely GabR from B. subtilis for which a significant amount of structural and functional information is available. The project includes two tasks: the first consists in the delineation of the molecular mechanism underlying the function of the GabR using molecular dynamics (MD) simulations; the second one is aimed at understanding the pathway through which the MocR subfamily has emerged during evolution.
A model of the entire GabR dimer complexed with the gamma-amino butyric acid (GABA), its effector, will be built using the GabR crystallographic structures available in the PDB. A long run of MD will be applied to the model to simulate the effect of GABA binding on the conformation of GabR.
Multiple sequence alignment, cluster and phylogenetic analysis, and homology modeling will be applied on a representative set of MocRs. This study should help delineating the MocR regulator evolution path and functional diversification which may have proceeded either through a single or subsequent multiple fusion events between an ancestral gene coding for a wHTH domain and one or different fold type-I PLP enzyme-coding genes.
The project points to study the detailed molecular mechanism underlying the response of the GabR-TR to the binding of GABA effector. Several authors suggested that most transcriptional regulators utilize a mechanism resembling that of the allosterism observed in many enzymes. Analysis of allosteric mechanisms however is often a challenging task requiring the application of a set of different and sophisticated approaches. For that reason studies specific for this particular aspect of the function of transcriptional regulators are still relatively scant. In our case, we intend to apply extensive molecular dynamics calculations to simulate the conformational movements of holo-GabR with and without GABA external aldimine. Dimensions of the protein systems are quite challenging since the entire dimer contains about 900 amino acid residues. Moreover, the dimer dynamics must be simulated in a volume of virtual water molecules and ions containing thousands of atoms. To obtain meaningful results we intend to extend the MD simulation to at least 1 microsecond time span, which is a remarkably long period for the size of the system under investigation.
We expect that the results will shed light on the molecular basis of the GabR conformational transitions induced by GABA binding. Moreover, the results will provide a framework and a guide for investigating the molecular mechanism of other regulators of the same or different families. The computational protocols which will be developed for trajectory analyses of such a large system may be extended and applied to the study of other regulators. For that reason the potential implications of the proposed research extends beyond the sole characterization of GabR.
Study of the evolution of the MocR will contribute to develop a theoretical scheme to investigate the mechanism through which gene fusion can evolve new proteins with new functions from existing ones. Many examples of proteins emerged during evolution through gene fusion are already known. Among these are several bacterial transcriptional regulators. However, the case of MocRs displays distinguishing features: the effector domain has a relatively large size compared with that of other effector/binding domains of different GntR regulators. Moreover, fold type-I PLP-dependent enzymes represent a protein family emerged through a complex evolution pathway which has lead to a wide functional diversification through conservation of an almost unaltered three-dimensional scaffold. Until the discover of the MocRs , fold type-I PLP dependent proteins were deemed to possess mainly catalytic activity. Several experimental and theoretical data strongly support the notion that the AAT-like domain of MocRs have lost most, if not all, the catalytic activity yet maintaining the ability to bind PLP and an effector molecule often resembling or being identical to the substrates of the enzyme counterparts. This research can also provide insights in the intricate evolutionary process of this ancient and widespread family of enzymes. Moreover, the study of the evolution and structural features of MocR-TRs can provide clues as how an enzyme can lose its catalytic activity to become a binding protein. Knowledge acquired from these analyses may contribute to develop protein engineering strategies able to modify the activity and function of existing enzymes.