Refinement of 3D models of horseradish peroxidase isoenzyme C: Predictions of 2D NMR assignments and substrate binding sites

Abstract

In this study, two alternative three-dimensional (3D) models of horseradish peroxidase (HRP-C)-differing mainly in the structure of a long untemplated insertion-were refined, systematically assessed, and used to make predictions that can both guide and be tested by future experimental studies. A key first step in the model-building process was a procedure for multiple sequence alignment based on structurally conserved regions and key conserved residues, including those side chains providing ligands to the two Ca2+ binding sites. The model refinements reported here include (1) optimization of side-chain conformations; (3) addition of structural waters using a template-independent procedure; (2) structural refinement of the untemplated 34 amino acid insertion located between the F and G helices, using both energy criteria and NMR data; (4) unconstrained energy optimization of the refined models. Using these procedures, two refined structures of HRP-C were obtained, differing mainly in the conformation of this long insertion. The presence of residues in this insertion that could potentially interact with bound substrates suggests a functional role that may be related to the general ability of class III peroxidases to form stable 1:1 complexes with a variety of substrates. The structural validity of the models was systematically assessed by a variety of criteria. Most notably, the ProsaII z scores and Profiles 3D scores of the two HRP-C models indicated that they are significantly better than would be obtained by simple amino acid replacement, using any of the known structures as a template. These two 3D HRP-C models, were then used to predict candidate residues for the assignment of NOESY cross-peaks previously noted in 2D-NMR studies. Specifically, the residues known as Ile X, Phe A, Phe B, aliphatic residue Q, and Ile T. Candidate substrate binding sites were also identified and compared with experimentally based predictions. This work is timely because new X-ray structures are anticipated that will facilitate the validation of these procedures.

Homology modeling and substrate binding study of human CYP2C9 enzyme

“…This initial model was refined in three steps: (1) relaxation of the side‐chain conformations by constrained energy optimization with only the side‐chain atoms allowed to move, (2) addition of structural waters using the in‐house program HYDRAS,64 and (3) unconstrained energy optimization of the entire system using the AMBER65 suite of programs. Structural waters were found to be essential to preserving the stability of the model.…”

Structural motifs as functional microdomains in G‐protein‐coupled receptors: Energetic considerations in the mechanism of activation of the serotonin 5‐HT2A receptor by disruption of the ionic lock of the arginine cage*

“…In turn, the inferences from the experiments are implemented in the 3D receptor models which can now be considered highly reliable if they incorporate the great variety of direct experimental data as well as results from various approaches ranging from computational genomics to biophysics and bioinformatics (for further discussion see 5). Gilda Loew's lab has contributed significantly to this library of 3D models of GPCRs and their analysis in mechanistic terms 7–19.…”

Homology modeling and substrate binding study of human CYP2C18 and CYP2C19 enzymes

“…During this procedure, the protein backbone was constrained not to move, so that movement occurred exclusively in the sidechain conformations. In the next stage, structural waters were added, using the in‐house program HYDRAS 22. These structural waters are comparable to the crystallographic waters found in protein X‐ray structures.…”