Sébastien Morin

A practical guide to protein dynamics from 15N spin relaxation in solution.

2010 Elsevier B.V. All rights reserved.

TEM-1 Backbone Dynamics—Insights from Combined Molecular Dynamics and Nuclear Magnetic Resonance

Dynamic properties of class A beta-lactamase TEM-1 are investigated from molecular dynamics (MD) simulations. Comparison of MD-derived order parameters with those obtained from model-free analysis of nuclear magnetic resonance (NMR) relaxation data shows high agreement for N-H moieties within alpha- and beta-secondary structures, but significant deviation for those in loops. This was expected, because motions slower than the protein global tumbling often take place in loop regions. As previously shown using NMR, TEM-1 is a highly ordered protein. Motions are observed within the Omega loop that could, upon substrate binding, stabilize E166 in a catalytically efficient position as the cavity between the protein core and the Omega loop is partially filled. The rigidity of active site residues is consistent with the enzyme high turnover number. MD data are also shown to be useful during the model selection step of model-free analysis: local N-H motions observed over the course of the trajectories help assess whether a peptide plan undergoes low or high amplitude motions on one or more timescales. This joint use of MD and NMR provides a better description of protein dynamics than would be possible using either technique alone.

Structure and dynamics of Mycobacterium tuberculosis truncated hemoglobin N: insights from NMR spectroscopy and molecular dynamics simulations.

The potent nitric oxide dioxygenase (NOD) activity (trHbN-Fe²⁺-O₂ + (•)NO → trHbN-Fe³⁺-OH₂ + NO₃⁻) of Mycobacterium tuberculosis truncated hemoglobin N (trHbN) protects aerobic respiration from inhibition by (•)NO. The high activity of trHbN has been attributed in part to the presence of numerous short-lived hydrophobic cavities that allow partition and diffusion of the gaseous substrates (•)NO and O₂ to the active site. We investigated the relation between these cavities and the dynamics of the protein using solution NMR spectroscopy and molecular dynamics (MD). Results from both approaches indicate that the protein is mainly rigid with very limited motions of the backbone N-H bond vectors on the picoseconds-nanoseconds time scale, indicating that substrate diffusion and partition within trHbN may be controlled by side-chains movements. Model-free analysis also revealed the presence of slow motions (microseconds-milliseconds), not observed in MD simulations, for many residues located in helices B and G including the distal heme pocket Tyr33(B10). All currently known crystal structures and molecular dynamics data of truncated hemoglobins with the so-called pre-A N-terminal extension suggest a stable α-helical conformation that extends in solution. Moreover, a recent study attributed a crucial role to the pre-A helix for NOD activity. However, solution NMR data clearly show that in near-physiological conditions these residues do not adopt an α-helical conformation and are significantly disordered and that the helical conformation seen in crystal structures is likely induced by crystal contacts. Although this lack of order for the pre-A does not disagree with an important functional role for these residues, our data show that one should not assume an helical conformation for these residues in any functional interpretation. Moreover, future molecular dynamics simulations should not use an initial α-helical conformation for these residues in order to avoid a bias based on an erroneous initial structure for the N-termini residues. This work constitutes the first study of a truncated hemoglobin dynamics performed by solution heteronuclear relaxation NMR spectroscopy.

Conservation of Flexible Residue Clusters among Structural and Functional Enzyme Homologues

Background: It remains unclear whether structural homologues rely on similar concerted motions to promote enzyme function. Results: Ribonuclease homologues display similar, contiguous clustering motions that can be modulated by mutagenesis. Conclusion: Conformational flexibility can be conserved between distant structural homologues. Significance: Controlling dynamics to modulate function has broad implications in protein engineering and allosteric drug design. Conformational flexibility between structural ensembles is an essential component of enzyme function. Although the broad dynamical landscape of proteins is known to promote a number of functional events on multiple time scales, it is yet unknown whether structural and functional enzyme homologues rely on the same concerted residue motions to perform their catalytic function. It is hypothesized that networks of contiguous and flexible residue motions occurring on the biologically relevant millisecond time scale evolved to promote and/or preserve optimal enzyme catalysis. In this study, we use a combination of NMR relaxation dispersion, model-free analysis, and ligand titration experiments to successfully capture and compare the role of conformational flexibility between two structural homologues of the pancreatic ribonuclease family: RNase A and eosinophil cationic protein (or RNase 3). In addition to conserving the same catalytic residues and structural fold, both homologues show similar yet functionally distinct clusters of millisecond dynamics, suggesting that conformational flexibility can be conserved among analogous protein folds displaying low sequence identity. Our work shows that the reduced conformational flexibility of eosinophil cationic protein can be dynamically and functionally reproduced in the RNase A scaffold upon creation of a chimeric hybrid between the two proteins. These results support the hypothesis that conformational flexibility is partly required for catalytic function in homologous enzyme folds, further highlighting the importance of dynamic residue sectors in the structural organization of proteins.

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

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.

Synergistic Applications of MD and NMR for the Study of Biological Systems

Modern biological sciences are becoming more and more multidisciplinary. At the same time, theoretical and computational approaches gain in reliability and their field of application widens. In this short paper, we discuss recent advances in the areas of solution nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics (MD) simulations that were made possible by the combination of both methods, that is, through their synergistic use. We present the main NMR observables and parameters that can be computed from simulations, and how they are used in a variety of complementary applications, including dynamics studies, model-free analysis, force field validation, and structural studies.

Simple tests for the validation of multiple field spin relaxation data.

Abstract15N spin relaxation data is widely used to extract detailed dynamic information regarding bond vectors such as the amide N–H bond of the protein backbone. Analysis is typically carried using the Lipari–Szabo model-free approach. Even though the original model-free equation can be determined from single field R1, R2 and NOE, over-determination of more complex motional models is dependent on the recording of multiple field datasets. This is especially important for the characterization of conformational exchange which affects R2 in a field dependent manner. However, severe artifacts can be introduced if inconsistencies arise between experimental setups with different magnets (or samples). Here, we propose the use of simple tests as validation tools for the assessment of consistency between different datasets recorded at multiple magnetic fields. Synthetic data are used to show the effects of inconsistencies on the proposed tests. Moreover, an analysis of data currently deposited in the BMRB is performed. Finally, two cases from our laboratory are presented. These tests are implemented in the open-source program relax, and we propose their use as a routine check-up for assessment of multiple field dataset consistency prior to any analysis such as model-free calculations. We believe this will aid in the extraction of higher quality dynamics information from 15N spin relaxation data.

Monocytes contribute to differential immune pressure on R5 versus X4 HIV through the adipocytokine visfatin/nampt

Background The immune system exerts a diversifying selection pressure on HIV through cellular, humoral and innate mechanisms. This pressure drives viral evolution throughout infection. A better understanding of the natural immune pressure on the virus during infection is warranted, given the clinical interest in eliciting and sustaining an immune response to HIV which can help to control the infection. We undertook to evaluate the potential of the novel HIV-induced, monocyte-derived factor visfatin to modulate viral infection, as part of the innate immune pressure on viral populations. Results We show that visfatin is capable of selectively inhibiting infection by R5 HIV strains in macrophages and resting PBMC in vitro, while at the same time remaining indifferent to or even favouring infection by X4 strains. Furthermore, visfatin exerts a direct effect on the relative fitness of R5 versus X4 infections in a viral competition setup. Direct interaction of visfatin with the CCR5 receptor is proposed as a putative mechanism for this differential effect. Possible in vivo relevance of visfatin induction is illustrated by its association with the dominance of CXCR4-using HIV in the plasma. Conclusions As an innate factor produced by monocytes, visfatin is capable of inhibiting infections by R5 but not X4 strains, reflecting a potential selective pressure against R5 viruses.

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

Enzyme engineering has been facilitated by recombination of close homologues, followed by functional screening. In one such effort, chimeras of two class-A β-lactamases – TEM-1 and PSE-4 – were created according to structure-guided protein recombination and selected for their capacity to promote bacterial proliferation in the presence of ampicillin (Voigt et al., Nat. Struct. Biol. 2002 9:553). To provide a more detailed assessment of the effects of protein recombination on the structure and function of the resulting chimeric enzymes, we characterized a series of functional TEM-1/PSE-4 chimeras possessing between 17 and 92 substitutions relative to TEM-1 β-lactamase. Circular dichroism and thermal scanning fluorimetry revealed that the chimeras were generally well folded. Despite harbouring important sequence variation relative to either of the two ‘parental’ β-lactamases, the chimeric β-lactamases displayed substrate recognition spectra and reactivity similar to their most closely-related parent. To gain further insight into the changes induced by chimerization, the chimera with 17 substitutions was investigated by NMR spin relaxation. While high order was conserved on the ps-ns timescale, a hallmark of class A β-lactamases, evidence of additional slow motions on the µs-ms timescale was extracted from model-free calculations. This is consistent with the greater number of resonances that could not be assigned in this chimera relative to the parental β-lactamases, and is consistent with this well-folded and functional chimeric β-lactamase displaying increased slow time-scale motions.

Combination of the CCL5-derived peptide R4.0 with different HIV-1 blockers reveals wide target compatibility and synergic cobinding to CCR5.

ABSTRACT R4.0, a synthetic CCL5/RANTES-derived peptide, exerts potent anti-HIV-1 activity via its nonactivating interaction with CCR5, the major HIV-1 coreceptor. CCR5 chronic activation may promote undesirable inflammatory effects and enhance viral infection; thus, receptor antagonism is a necessary requisite. HIV-1 gp120, CCL5, and maraviroc dock on CCR5 by sharing two receptor sites: the N terminus and the second extracellular loop. In combination studies, R4.0, CCL5, and maraviroc exhibited concomitant interactions with CCR5 and promoted synergic inhibition of HIV-1 in acute-infection assays. Furthermore, various degrees of additive/synergic HIV-1 inhibition were observed when R4.0 was tested in combination with drugs and lead compounds directed toward different viral targets (gp120, gp41, reverse transcriptase, and protease). In combination with tenofovir, R4.0 provides cross-clade synergic inhibition of primary HIV-1 isolates. Remarkably, an in vitro-generated maraviroc-resistant R5 HIV-1 strain was inhibited by R4.0 comparably to the wild-type strain, suggesting the presence of viral resistance barriers similar to those reported for CCL5. Overall, R4.0 appears to be a promising lead peptide with potential for combination in anti-HIV-1 therapy and in microbicide development to prevent sexual HIV-1 transmission.