Abstract The backbone dynamics and overall tumbling of protein G have been investigated using 15 N relaxation. Comparison of measured R 2 / R 1 relaxation rate ratios with known three‐dimensional coordinates of the protein show that the rotational diffusion tensor is significantly asymmetric, exhibiting a prolate axial symmetry. Extensive Monte Carlo simulations have been used to estimate the uncertainty due to experimental error in the relaxation rates to be D∥︁/D 1 = 1.68± 0.08, while the dispersion in the NMR ensemble leads to a variation of Dn/D 1 = 1.65 ± 0.03. Incorporation of this tensorial description into a Lipari‐Szabo type analysis of internal motion has allowed us to accurately describe the local dynamics of the molecule. This analysis differs from an earlier study where the overall rotational diffusion was described by a spherical top. In this previous analysis, exchange parameters were fitted to many of the residues in the alpha helix. This was interpreted as reflecting a small motion of the alpha helix with respect to the beta sheet. We propose that the differential relaxation properties of this helix compared to the beta sheet are due to the near‐orthogonality of the NH vectors in the two structural motifs with respect to the unique axis of the diffusion tensor. Our analysis shows that when anisotropic rotational diffusion is taken into account NH vectors in these structural motifs appear to be equally rigid. This study underlines the importance of a correct description of the rotational diffusion tensor if internal motion is to be accurately investigated.
Abstract In the context of NMR, ‘ chemical exchange’ refers to any process in which a nucleus exchanges between two or more environments in which its NMR parameters, (e.g. chemical shift, scalar coupling, or relaxation) differ. These may be intramolecular or intermolecular processes.
β-Strand mediated protein-protein interactions (PPIs) represent underexploited targets for chemical probe development despite representing a significant proportion of known and therapeutically relevant PPI targets. β-strand mimicry is challenging given that both amino acid side-chains and backbone hydrogen-bonds are typically required for molecular recognition, yet these are oriented along perpendicular vectors. This paper describes an alternative approach using GKAP/SHANK1 PDZ as a model and dynamic ligation screening to identify small-molecule replacements for tranches of peptide sequence. A peptide truncation of GKAP functionalized at the N- and C-termini with acylhydrazone groups was used as an anchor. Reversible acylhydrazone bond exchange with a library of aldehyde fragments in the presence of the protein as template and <i>in situ</i> screening using a fluorescence anisotropy (FA) assay identified peptide hybrid hits with comparable affinity to the GKAP peptide binding sequence. Identified hits were validated using FA, ITC, NMR and X-ray crystallography to confirm selective inhibition of the target PDZ-mediated PPI and mode of binding. These analyses together with molecular dynamics simulations demonstrated the ligands make transient interactions with an unoccupied basic patch through electrostatic interactions, establishing proof-of-concept that this unbiased approach to ligand discovery represents a powerful addition to the armory of tools that can be used to identify PPI modulators.
MUPs (major urinary proteins) play an important role in chemical signalling in rodents and possibly other animals. In the house mouse (Mus musculus domesticus) MUPs in urine and other bodily fluids trigger a range of behavioural responses that are only partially understood. There are at least 21 Mup genes in the C57BL/6 mouse genome, all located on chromosome 4, encoding sequences of high similarity. Further analysis separates the MUPs into two groups, the 'central' near-identical MUPs with over 97% sequence identity and the 'peripheral' MUPs with a greater degree of heterogeneity and approximately 20-30% non-conserved amino acids. This review focuses on differences between the two MUP sub-groups and categorizes these changes in terms of molecular structure and pheromone binding. As small differences in amino acid sequence can result in marked changes in behavioural response to the signal, we explore the potential of single amino acid changes to affect chemical signalling and protein stabilization. Using analysis of existing molecular structures available in the PDB we compare the chemical and physical properties of the ligand cavities between the MUPs. Furthermore, we identify differences on the solvent exposed surfaces of the proteins, which are characteristic of protein-protein interaction sites. Correlations can be seen between molecular heterogeneity and the specialized roles attributed to some MUPs.
It is normal for a biological system to be in a dynamic state. The function of many biological systems depends on their flexibility, and here NMR can provide the experimental basis for investigating the mechanical function of such systems. The types of motions that are thought to occur in proteins, their frequency ranges, and the methods for their detection are summarized in Table 1. It is important to distinguish ubiquitous thermal vibrations or group rotations from the more extensive motions that can be propagated through larger segments of a structure. Motions and dynamics are reflected by five different NMR parameters: chemical shift, spin-spin coupling constant, the area enclosed by a resonance, relaxation time, and nuclear Overhauser effect (1). The NMR data can be used to provide either qualitative evidence of flexibility or quantitative measurements of exchange rates.
Abstract ZL006 and IC87201 have been presented as efficient inhibitors of the nNOS/PSD-95 protein-protein interaction and shown great promise in cellular experiments and animal models of ischemic stroke and pain. Here, we investigate the proposed mechanism of action of ZL006 and IC87201 using biochemical and biophysical methods, such as fluorescence polarization (FP), isothermal titration calorimetry (ITC) and 1 H- 15 N HSQC NMR. Our data show that under the applied in vitro conditions, ZL006 and IC87201 do not interact with the PDZ domains of nNOS or PSD-95, nor inhibit the nNOS-PDZ/PSD-95-PDZ interface by interacting with the β-finger of nNOS-PDZ. Our findings have implications for further medicinal chemistry efforts of ZL006, IC87201 and analogues and challenge the general and widespread view on their mechanism of action.
<div>Abstract<p>The nuclear deubiquitylase BRCA1-associated protein 1 (BAP1) is frequently inactivated in malignant pleural mesothelioma (MPM) and germline <i>BAP1</i> mutation predisposes to cancers including MPM. To explore the influence on cell physiology and drug sensitivity, we sequentially edited a predisposition mutation (<i>w-</i>) and a promoter trap (<i>KO</i>) into human mesothelial cells. <i>BAP1<sup>w-/KO</sup></i> MeT5A cells express less BAP1 protein and phenocopy key aspects of BAP1 loss in MPM. Stable isotope labeling with amino acids in cell culture–mass spectrometry revealed evidence of metabolic adaptation, with concomitant alteration of cellular metabolites. In MeT5A, BAP1 deficiency reduces glycolytic enzyme levels but increases enzymes involved in the tricarboxylic acid cycle and anaplerotic pathways. Notably both argininosuccinate synthase 1 (ASS1), essential for cellular synthesis of arginine, and its substrate aspartate, are elevated in <i>BAP1<sup>w-/KO</sup></i> MeT5A cells. Likewise, ASS1 expression is higher in BAP1-altered MPM cell lines, and inversely correlates with BAP1 in The Cancer Genome Atlas MESO dataset. Elevated ASS1 is also evident by IHC staining in epithelioid MPM lacking nuclear BAP1 expression, with improved survival among patients with BAP1-negative/ASS1-expressing tumors. Alterations in arginine metabolism may sensitize cells to metabolic drugs and we find that BAP1-negative/ASS1-expressing MPM cell lines are more sensitive to ASS1 inhibition, although not to inhibition of purine synthesis by mizoribine. Importantly, <i>BAP1<sup>w-/KO</sup></i> MeT5A become desensitized to arginine deprivation by pegylated arginine deiminase (ADI-PEG20), phenocopying BAP1-negative/ASS1-expressing MPM cell lines.</p>Implications:<p>Our data reveal an interrelationship between BAP1 and arginine metabolism, providing a potential means of identifying patients with epithelioid MPM likely to benefit from ADI-PEG20.</p></div>
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