Exploring Active Site Communication in Malate Dehydrogenase: The Role of First and Second Sphere Residues

Abstract

Novel therapeutic interventions for bacterial diseases have been of interest due to the rise in infections over the last decade. Much recent focus of drug design has been on “Allosteric” drugs targeting either existing or cryptic‐allosteric sites on proteins. Development of effective drugs depends in part on understanding fundamental features of protein structure and function, including binding site specificity and molecular mechanisms of allostery, including the transmission of signals from binding site to active site, a process that often involves relay of information across subunit interfaces. While a binding site contains “first sphere” residues (those touching the ligand) the transmission of information requires the involvement of “second sphere” residues. Second sphere residues may also govern the local polarity of first sphere residues contributing to binding and/or catalysis as well as providing triggers for subunit interactions. Glyoxysomal Malate Dehydrogenase (gMDH) catalyzes the reversible oxidation/reduction of substrates malate/oxaloacetate, coupled with cofactor conversion NAD+/NADH, a key stage in the Tricarboxylic Acid Cycle. MDH is known to be regulated via citrate and substrate inhibition with both processes thought to involve allosteric subunit communication. The active site contains a number of conserved. first sphere residues that contribute to catalysis and substrate binding including 3 conserved Arginines (R124, R130, R196) and the His‐Asp dyad (H220, D193) involved in catalysis.. To identify potential second sphere residues, High‐quality Protein Interactomes (HINT) tables were utilized to analyze interactions of H220 and D193 with nearby residues. HINT tables provide the types and magnitude of interactions occurring. HINT analysis was conducted on MDH with no ligand bound or with substrates This analysis identified four residues, V194, G218, Q251 and M271 as potential second sphere residues that might contribute to subunit interactions. Potential key interactions identified include V194 with D193 (active site), G218 with H220 (active site), Q251 with T255 (located on the interface loop containing S266). And M271 with close neighbor residues 272–276 and, when citrate is bound, across the subunit interface with D87 which is connected to the active site on the opposite subunit. Four mutants, V194E, G218W, M271Q and Q251A were constructed, using Quikchange mutagenesis, expressed and purified by NiNTA Chromatography. Mutants were characterized specific activity, by CD and Fluorescence based Thermal Shift assays to assess structure, stability and cofactor or citrate binding, and by kinetics assays to assess oxaloacetate and NADH interaction. All mutations resulted in altered specific activity. The Q251A mutation decreased Km for NADH while M271Q resulted in an increase in Km for NADH. With prior results on subunit interactions these observations give rise to a model of cofactor and substrate induced alterations at the subunit interface mediated by active site interactions and second sphere resi...