Biology subjects

Milicaj, J.

Publications and source records attributed to Milicaj, J..

3 recordsLinked to original sources

bioRxiv2022

Kinetic Characterization and Computational Modeling of the Escherichia coli Heptosyltransferase II: Exploring the Role of Protein Dynamics in Catalysis for a GT-B Glycosyltransferase

Glycosyltransferases (GTs) are enzymes that are uniquely adapted to promote the formation of a glycosidic bond between a sugar molecule and a wide variety of substrates. Heptosyltransferase II (HepII) is a GT involved in the lipopolysaccharide (LPS) biosynthetic pathway that transfers the seven-carbon sugar (L-glycero-D-manno-heptose; Hep) onto a lipid anchored glycopolymer (heptosylated Kdo2-Lipid A, Hep-Kdo2-Lipid A or HLA). LPS plays a key role in Gram-negative bacterial sepsis as a stimulator of the human immune response and has been used as an adjuvant in vaccines. As such, ongoing efforts towards inhibition of LPS biosynthetic enzymes to aid development of novel antimicrobial therapeutics has driven significant effort towards the characterization of these enzymes. Three heptosyltransferases are involved in the inner-core biosynthesis, with E. coli HepII being the last to be quantitatively characterized in vivo, as described herein. HepII shares modest sequence similarity with heptosyltransferase I (HepI) while maintaining a high degree of structural homology. Here we report the first kinetic and biophysical characterization of HepII and demonstrate the properties of HepII that are shared by HepI to include sugar donor promiscuity, and sugar acceptor induced secondary structural changes which results in significant thermal stabilization. HepII also has an increased catalytic efficiency and a significantly tighter binding affinity for both of its substrates, with an insensitivity to the number of acyl chains on the sugar acceptor. Additionally, a structural model of the HepII ternary complex, refined by molecular dynamics simulations, was developed to probe potentially important substrate-protein contacts and revealed the potential of Tryptophan (Trp) residues responsible for reporting on ligand binding. As was previously described for HepI, Tryptophan fluorescence in HepII allowed observation of substrate induced changes in Trp fluorescence intensity which enabled determination of substrate dissociation constants. Combined, these efforts meaningfully enhance our understanding of the Heptosyltransferase family of enzymes and will aid in future efforts to design novel, potent and specific inhibitors for this family of enzymes.

biochemistry↗

bioRxiv2021

Ligand Induced Conformational and Dynamical Changes in a GT-B Glycosyltransferase:Molecular Dynamic Simulations of Heptosyltransferase I Apo, Binary and Ternary Complexes

Understanding the dynamical motions and ligand recognition motifs of specific glycosyltransferase enzymes, like Heptosyltransferase I (HepI), is critical to discerning the behavior of other carbohydrate binding enzymes. Prior studies in our lab demonstrated that glycosyltransferases in the GT-B structural class, which are characterized by their connection of two Rossman-like domains by a linker region, have conservation of both structure and dynamical motions, despite low sequence conservation, therefore making discoveries found in HepI transferable to other GT-B enzymes. Through a series of 100 nanosecond Molecular Dynamics simulations of HepI in apo enzyme state, and also in the binary and ternary complexes with the native substrates/products. Ligand free energy analysis allowed determination of an anticipated enzymatic path for ligand binding and release. Principle component, dynamic cross correlation and network analyses of the simulation trajectories revealed that there are not only correlated motions between the N- and C-termini, but also that residues within the N-terminal domain communicate via a path that includes substrate proximal residues of the C-terminal domain. Analysis of structural changes, energetics of substrate/products binding and changes in pKa have elucidated a variety of inter- and intradomain interactions that are critical for catalysis. These data corroborate and allow visualization of previous experimental observations of protein conformational changes of HepI. This study has provided valuable insights into the regions involved in HepI conformational rearrangement upon ligand binding, and are likely to enhance efforts to develop new dynamics disrupting enzyme inhibitors for GT-B structural enzymes in the future.

biochemistry↗

bioRxiv2020

Conserved conformational hierarchy across functionally divergent glycosyltransferases of the GT-B structural superfamily as determined from microsecond molecular dynamics

It has long been understood that some proteins to undergo conformational transitions enroute to the Michaelis Complex to allow chemistry. Examination of crystal structures of glycosyltransferase enzymes in the GT-B structural class reveals that the presence of ligand in the active site is necessary for the protein to crystalize in the closed conformation. Herein we describe microsecond molecular dynamics simulations of two evolutionarily unrelated glycosytransferases, HepI and GtfA. Simulations were performed using these proteins in the open and closed conformations, (respectively,) and we sought to identify the major dynamical modes and communication networks which allow conformational transition between the open and closed structures. We provide the first reported evidence (within the scope of our experimental parameters) that conformational hierarchy/directionality of the interconversion between open and closed conformations is a conserved feature of enzymes of the same structural superfamily. Additionally, residues previously identified to be important for substrate binding in HepI were shown to have strong negative correlations with non-ionizable residues distal to the active site. Mutagenesis of these residues produced mutants with altered enzymatic efficiency exhibiting lower Km values, while the kcat is effectively unchanged. The negatively correlated motions of these residues are important for substrate binding and forming the Michaelis complex, without impacting the activation barrier for catalysis. This suggests that in the bi-domain HepI, the enzyme dynamics did not impact the transition state stabilization or chemistry, but rather earlier steps along the reaction coordinate, leading to the reorganization of the active site electrostatic environment required for catalysis.Competing Interest StatementThe authors have declared no competing interest.AbbreviationsGTglycosyltransferaseHepIheptosyltransferase IheptoseL-glycero-D-manno-heptoseADP-heptose or ADPHADP-L-glycero-D-manno-heptoseLPSlipopolysaccharide;ODLAO-deacylated E. coli Kdo2-lipid A;Trptryptophan;Proproline;Glyglycine;Argarginine;Lyslysine;CDcircular dichroism;MDMolecular Dynamics;PDBProtein Data BankPCAPrincipal Component Analysis,DCCDynamic Cross CorrelationView Full Text

biophysics↗