With multi-drug resistant cases of tuberculosis increasing globally, better antibiotic drugs and novel drug-targets are becoming an urgent need. Traditional β-lactam antibiotics that disrupt the D,D-transpeptidases are not effective against mycobacteria, in part because mycobacteria rely mostly on β-lactam insensitive L,D-transpeptidases for biosynthesis and maintenance of their peptidoglycan layer. This reliance plays a major role in drug-resistance and persistence of Mycobacterium tuberculosis (Mtb) infections. The crystal structure at 1.7 Å resolution of the Mtb L,D-transpeptidase LdtMt2 containing a bound peptidoglycan fragment, reported here, provides information about catalytic site organization as well as substrate recognition by the enzyme. Based on our structural, kinetic, and calorimetric data, we propose a catalytic mechanism for LdtMt2 in which both acyl-acceptor and acyl-donor substrates reach the catalytic site from the same, rather than different, entrances. Together, this information provides vital insights for the development of novel drugs targeting this validated yet unexploited enzyme.

Conserved residues in protein-protein interfaces correlate with residue hot-spots. To obtain insight into their roles, we have studied their mobility. We have performed 39 explicit solvent simulations of 15 complexes and their monomers, with the interfaces varying in size, shape, and function. The dynamic behavior of conserved residues in unbound monomers illustrates significantly lower flexibility as compared to their environment, suggesting that already before binding they are constrained in a boundlike configuration. To understand this behavior, we have analyzed the inter- and intrachain hydrogen-bond residence-time in the interfaces. We find that conserved residues are not involved significantly in hydrogen bonds across the interface as compared to nonconserved. However, the monomer simulations reveal that conserved residues contribute dominantly to hydrogen-bond formation before binding. Packing of conserved residues across the trajectories is significantly higher before and after the binding, rationalizing their lower mobility. Backbone torsional angle distributions show that conserved residues assume restricted regions of space and the most visited conformations in the bound and unbound trajectories are similar, suggesting that conserved residues are preorganized. Combined with previous studies, we conclude that conserved residues, hot spots, anchor, and interface-buried residues may be similar residues, fulfilling similar roles.

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