Crystallographic Study of Peptidoglycan Biosynthesis Enzyme MurD: Domain Movement Revisited

Abstract

The biosynthetic pathway of peptidoglycan, an essential component of bacterial cell wall, is a well-recognized target for antibiotic development. Peptidoglycan precursors are synthesized in the bacterial cytosol by various enzymes including the ATP-hydrolyzing Mur ligases, which catalyze the stepwise addition of amino acids to a UDP-MurNAc precursor to yield UDP-MurNAc-pentapeptide. MurD catalyzes the addition of D-glutamic acid to UDP-MurNAc-L-Ala in the presence of ATP; structural and biochemical studies have suggested the binding of the substrates with an ordered kinetic mechanism in which ligand binding inevitably closes the active site. In this work, we challenge this assumption by reporting the crystal structures of intermediate forms of MurD either in the absence of ligands or in the presence of small molecules. A detailed analysis provides insight into the events that lead to the closure of MurD and reveals that minor structural modifications contribute to major overall conformation alterations. These novel insights will be instrumental in the development of new potential antibiotics designed to target the peptidoglycan biosynthetic pathway.

Structure of the essential peptidoglycan amidotransferase MurT/GatD complex from Streptococcus pneumoniae

“…ATP and lipid II docked on the central-domain of the crystal structure do not interact with the C-terminal domain. Both domains are known to move relative to each other in Mur ligases 26 , 36 , and an acidic residue on a loop of the C-terminal domain is hydrogen-bonded to the ATP ribose in the closed conformation. The corresponding loop of MurT harbors three consecutive conserved residues Lys321-Asn322-Pro323, and MT Asn322 may contact the ATP ribose if the domains are brought in proximity.…”

“…The corresponding loop of MurT harbors three consecutive conserved residues Lys321-Asn322-Pro323, and MT Asn322 may contact the ATP ribose if the domains are brought in proximity. Reorientation of the domains may occur when the hinge MT Arg304 interacts with ATP 36 .…”

Structural conservation of the PIN domain active site across all domains of life

“…A . Left , structure of E. coli MurD ligase (PDB 5A5F), an example of the HUP fold, which is a minimal version of the PIN domain fold . The bound molecule of uridine‐5'‐diphosphate‐ N ‐acetylmuramoyl‐l‐alanine (UMA) is shown with sticks; right , the PIN domain of M. tuberculosis VapC‐15 (PDB 4CHG) aligned to the HUP fold showing that the active site of these Rossmann‐like folds are located in similar places at the C‐terminal end of the parallel β‐strands .…”

Learning protein conformational space by enforcing physics with convolutions and latent interpolations

“…The protein consists of three globular domains, one of which (residues 299-437) undergoes a large scale rearrangement (from open to closed state) triggered by substrate binding to activate the catalytic site. The open (PDB: 1E0D [23]) and closed (PDB: 3UAG [24]) states, as well as a few intermediates (PDB: 5A5E and 5A5F [25]), have been crystallized, providing key experimental evidence about the possible protein's mode of action. MD simulations of the open and closed states, and of the transition between them have been previously carried out [19], providing a useful dataset for our network training and its performance evaluation.…”

“…MurD has been crystallized in its closed (PDB: 3UAG [24]) and open (PDB: 1E0D [23]) states, as well as in intermediates between the two (PDB: 5A5E and 5A5F [25] with a backbone RMSD of secondary structure elements equal to 1.12Å between them). The difference between these states comes from large scale conformational change of one of its three globular domains (residues 299-437) caused by substrate binding.…”