NMR Dynamics of PSE-4 β-Lactamase: An Interplay of ps-ns Order and μs-ms Motions in the Active Site

Abstract

The backbone dynamics for the 29.5 kDa class A beta-lactamase PSE-4 is presented. This solution NMR study was performed using multiple field (15)N spin relaxation and amide exchange data in the EX2 regime. Analysis was carried out with the relax program and includes the Lipari-Szabo model-free approach. Showing similarity to the homologous enzyme TEM-1, PSE-4 is very rigid on the ps-ns timescale, although slower mus-ms motions are present for several residues; this is especially true near the active site. However, significant dynamics differences exist between the two homologs for several important residues. Moreover, our data support the presence of a motion of the Omega loop first detected using molecular dynamics simulations on TEM-1. Thus, class A beta-lactamases appear to be a class of highly ordered proteins on the ps-ns timescale despite their efficient catalytic activity and high plasticity toward several different beta-lactam antibiotics. Most importantly, catalytically relevant mus-ms motions are present in the active site, suggesting an important role in catalysis.

Chimeric β-Lactamases: Global Conservation of Parental Function and Fast Time-Scale Dynamics with Increased Slow Motions

“…Average R 1 , R 2 , and { 1 H}- 15 N NOE for cTEM-17m are shown in Table 4 along with previously reported values for the TEM-1 and PSE-4 parental proteins [23], [24]. The average backbone relaxation parameters for cTEM-17m were comparable to TEM-1 and PSE-4 at both fields studied.…”

“…To eliminate the potential effect of sample or field homogeneity degradation over time on measured exponential decays, relaxation delays were acquired in an interleaved manner [23], [29]. All NMR data were processed using NMRPipe/NMRDraw [39], peak amplitudes were measured in NMRView [40], and fits performed as previously described [24]. A consistency test of multiple field transverse relaxation data was performed within “relax” (version 1.2.14) [41], [42] as described earlier [24], [43].…”

“…Dual optimization of the model-free parameters and the global diffusion tensor was done according to d’Auvergne and Gooley [41], with modifications as in [24] (see below). Only residues for which data was available at the two magnetic fields (500 and 600 MHz) were analyzed.…”

“…The best model for each residue was then selected using the AICc (Akaike Information Criteria for small sample size) approach to data fitting, to prevent overfitting. Errors on the extracted local model-free parameters were obtained from 500 Monte Carlo simulations as previously described [24].…”

15N, 13C and 1H backbone resonance assignments of an artificially engineered TEM-1/PSE-4 class A β-lactamase chimera and its deconvoluted mutant

“…Its structure, kinetic parameters and dynamics have been studied by X-ray crystallography, molecular dynamics simulations and NMR (Minasov et al 2002; Savard and Gagné 2006). Its homolog, PSE-4, was also characterized by NMR and X-ray crystallography to report its structure, dynamics and kinetics (Lim et al 2001; Morin and Gagné 2009). TEM-1 and PSE-4 share moderate sequence identity (40 %) and conserved folds (backbone RMSD 0.99 Å).…”