The focus of this project was to explore Structure Function relationships in the enzyme UDP‐GlcNAc O transferase protein and design and build a physical model that illustrates key functional features of the protein. In particular our focus is on the Cross‐Talk between O‐GlcNAcylation and Phosphorylation that plays a critical role in overall regulation. Using the pdb file 4gyy.pdb from the paper “Crystal structure of human O‐GlcNAc Transferase with UDP‐5SGlcNAc and a peptide substrate” The structure illustrates the binding sites for both UDPGlcNAc and the peptide YPGGSTPVSSANMM with the requisite PV prior to the GlcNAcylatable S. Recent work by Leney et al (Proc Natl Acad Sci U S A. 2017 Aug 29;114(35):E7255–E7261) has shown that the preceding T can be phosphorylated and that the sequence TPVS is common in proteins thought to undergo GlcNAc‐Phosphorylation cross talk. We have also used computational models to create and built models of the peptide phosphorylated at the N‐3 Threonine. These models clearly illustrate that the phosphate group clashes with the U in UDP‐GlcNAC. Since the kinetic mechanism of OGT is ordered Bi‐Bi with UDPGlcNAc as the obligate first substrate, the phosphorylated peptide can no longer bind in the peptide substrate pocket explaining why excludes GlcNAcylation. The models described here can be used to illustrate three different aspects contained in the ASBMB 8 Core Concepts of Macromolecular Structure and Function: #3. Structure and function are related, #4. Macromolecular interactions, and #6. The biological activity of macromolecules is often regulated. In addition construction and use of the models illustrates the use of core concept #8, A variety of experimental and computational approaches can be used to observe and quantitatively measure the structure, dynamics and function of biological macromolecule.Support or Funding InformationFunded in part by NSF‐DUE 1725940 for the CREST Project.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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...

Malate Dehydrogenase (MDH) catalyzes the oxidation/reduction of malate/oxaloacetate through coupling with NAD+/NADH conversion, a key area in cellular metabolism and has been shown to be regulated by citrate. MDH is a homo‐dimer and x‐ray structures show a flexible loop, which upon substrate or citrate binding induces a closed conformational in one subunit, while the other remains open. This implies an as of yet unknown means of communication between the two subunits to convey the open/closed loop response. Concurrent work in the Bell Lab has shown two mutations, S266A & L269A, located in the active site and interface regions display significant changes in kinetic parameters. To further probe these regions and assess individual residue contributions to subunit communication several additional point mutants (I88A, K261M/Q, S270A/C, T255E/V) located between the active site and the subunit interface have been constructed, expressed, purified and characterized. The I88A mutant protein has a 10,000 fold decrease in Vmax while that of the T255E mutant is decreased 1,000 fold. Km for NADH is significantly increased in the I88A mutant while that for T255E remains unchanged. This suggests that decreased affinity for NADH in the I88A mutant results from disruption of the subunit interface., I88 is thought to interact with L269 on the opposite chain, while T255 may be involved in oxaloacetate/malate/citrate binding at the active site. This data is consistent with predicted effects of interface and active site mutations and contributes to a detailed understanding of a predicted inter‐subunit relay system to induce asymmetric conformational changes and ligand binding in this homo‐dimeric enzyme.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.