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Many metabolites are generated in one step of a biochemical pathway and consumed in a subsequent step. Such metabolic intermediates are often reactive molecules which, if allowed to freely diffuse in the intracellular milieu, could lead to undesirable side reactions and even become toxic to the cell. Therefore, metabolic intermediates are often protected as protein-bound species and directly transferred between enzyme active sites in multi-function al enzymes, multi-enzyme complexes, and metabolons. Sequestration of reactive metabolic intermediates thus con tributes to metabolic efficiency. It is not known, however, whether this evolutionary adaptation can be relaxed in response to challenges to organismal survival. Here, we report evolutionary repair experiments on Escherichia coli cells in which an enzyme crucial for the biosynthesis of proline has been deleted. The deletion makes cells unable to grow in a culture medium lacking proline. Remarkably, however, cell growth is efficiently restored by many single mutations (12 at least) in the gene of glutamine synthetase. The mutations cause the leakage to the intracellular milieu of a highly reactive phosphorylated intermediate common to the biosynthetic pathways of glutamine and pro line. This intermediate is generally assumed to exist only as a protein-bound species. Nevertheless, its diffusion upon mutation-induced leakage enables a new route to proline biosynthesis. Our results support that leakage of seques tered metabolic intermediates can readily occur and contribute to organismal adaptation in some scenarios. Enhanced availability of reactive molecules may enable the generation of new biochemical pathways and the poten tial of mutation-induced leakage in metabolic engineering is noted, Human Frontier Science Program RGP0041/2017, Spanish Government RTI2018-097142-B-100, Ministry of Science and Innovation, Spain (MICINN) 80NSSC18K1277, European Commission, Junta de Andalucia, Regional Andalusian Government E-BIO-464-UGR-20 2020_DOC_00541

We showcase the combination of experimental neutron scattering data and molecular dynamics (MD) simulations for exemplary phospholipid membrane systems. Neutron and X-ray reflectometry and small-angle scattering measurements are determined by the scattering length density profile in real space, but it is not usually possible to retrieve this profile unambiguously from the data alone. MD simulations predict these density profiles, but they require experimental control. Both issues can be addressed simultaneously by cross-validating scattering data and MD results. The strengths and weaknesses of each technique are discussed in detail with the aim of optimizing the opportunities provided by this combination.

Quantifying phospholipid bilayer-small molecule interactions is vital to the development of new drug candidates and/or medicinal therapies. However, obtaining these data remains problematic. Herein, we detail a phospholipid nanodisc assay which enables the elucidation of these interactions using conventional solution state NMR spectroscopy techniques.

The CLIC family of proteins display the unique feature of altering their structure from a soluble form to a membrane-bound chloride channel. CLIC1, a member of this family, can be found in the cytoplasm or in nuclear, ER and plasma membranes, with membrane overexpression linked to tumour proliferation. The molecular switch promoting CLIC1 membrane insertion has been related to environmental factors, but still remains unclear. Here, we use solution NMR studies to confirm that both the soluble and membrane bound forms are in the same oxidation state. Our data from fluorescence assays and chloride efflux assays indicate that Ca2+and Zn2+trigger association to the membrane into active chloride channels. We use fluorescence microscopy to confirm that an increase of the intracellular Ca2+leads to re-localisation of CLIC1 to both plasma and internal membranes. Finally, we show that soluble CLIC1 adopts an equilibrium of oligomeric species, and Ca2+/Zn2+mediated membrane insertion promotes the formation of a tetrameric assembly. Thus, our results identify Ca2+and Zn2+binding as the molecular switch promoting CLIC1 membrane insertion.SIGNIFICANCE STATEMENTCLIC1, a member of the CLIC family of proteins, is expressed as a soluble protein in cells but can insert in the membrane forming a chloride channel. This chloride channel form is upregulated in different types of cancers including glioblastoma and promote tumour invasiveness and metastasis. The factors promoting CLIC1 membrane insertion nor the mechanism of this process are yet understood. Here, we use a combination of solution NMR, biophysics and fluorescence microscopy to identify Ca2+and Zn2+binding as the switch to promote CLIC1 insertion into the membrane to form active chloride channels. We also provide a simple mechanism how such transition to the membrane occurs. Such understanding will enable subsequent studies on the structure of the chloride channel form and its inhibition.

The Sigma-1 Receptor (S1R) is a small, ligand-regulated integral membrane protein involved in cell homeostasis and the cellular stress response. The receptor has a multitude of protein and small molecule interaction partners with therapeutic potential. Newly reported structures of the human S1R in ligand-bound states provides essential insights into small molecule binding in the context of the overall protein structure. The structure also raises many interesting questions and provides an excellent starting point for understanding the molecular tricks employed by this small membrane receptor to modulate a large number of signaling events. Here, we review insights from the structures of ligand-bound S1R in the context of previous biochemical studies and propose, from a structural viewpoint, a set of important future directions.

In this chapter, we describe how NMR chemical shift titrations can be used to study the interaction between two proteins with emphasis on mapping the interface of the complex and determining the bind- ing affinity from a quantitative analysis of the experimental data. In particular, we discuss the appearance of NMR spectra in different chemical exchange regimes (fast, intermediate, and slow) and how these regimes affect NMR data analysis.

Background: Sigma-1 receptor is a ligand-regulated membrane protein chaperone involved in BiP regulation and the ER stress response. Results: The chaperone domain of human sigma-1 receptor is mostly helical with short extended regions. Conclusion: Regions of the sigma-1 receptor chaperone domain implicated in ligand and cholesterol binding can be mapped to separate helices. Significance: A structural framework for delineating sigma-1 receptor BiP and ligand interactions is presented., The sigma-1 receptor (S1R) is a ligand-regulated membrane protein chaperone involved in the ER stress response. S1R activity is implicated in diseases of the central nervous system including amnesia, schizophrenia, depression, Alzheimer disease, and addiction. S1R has been shown previously to regulate the Hsp70 binding immunoglobulin protein (BiP) and the inositol triphosphate receptor calcium channel through a C-terminal domain. We have developed methods for bacterial expression and reconstitution of the chaperone domain of human S1R into detergent micelles that enable its study by solution NMR spectroscopy. The chaperone domain is found to contain a helix at the N terminus followed by a largely dynamic region and a structured, helical C-terminal region that encompasses a membrane associated domain containing four helices. The helical region at residues ∼198–206 is strongly amphipathic and proposed to anchor the chaperone domain to micelles and membranes. Three of the helices in the C-terminal region closely correspond to previously identified cholesterol and drug recognition sites. In addition, it is shown that the chaperone domain interacts with full-length BiP or the isolated nucleotide binding domain of BiP, but not the substrate binding domain, suggesting that the nucleotide binding domain is sufficient for S1R interactions.

Protein-protein interactions occur with a wide range of affinities from tight complexes characterized by femtomolar dissociation constants to weak, and more transient, complexes of millimolar affinity. Many of the weak and transiently formed protein-protein complexes have escaped characterization due to the difficulties in obtaining experimental parameters that report on the complexes alone without contributions from the unbound, free proteins. Here, we review recent developments for characterizing the structures of weak protein-protein complexes using nuclear magnetic resonance spectroscopy with special emphasis on the utility of residual dipolar couplings.

Highlights • The second transmembrane domain (TM2) of S1R includes residues 91–107. • The S1R cytosolic region contains three α-helices. • The chaperone domain structure is largely unperturbed by the addition of TM2., The sigma-1 receptor (S1R) is a ligand-regulated membrane chaperone protein associated with endoplasmic reticulum stress response, and modulation of ion channel activities at the plasma membrane. We report here a solution NMR study of a S1R construct (S1R(Δ35)) in which only the first transmembrane domain and the eight-residue N-terminus have been removed. The second transmembrane helix is found to be composed of residues 91–107, which corresponds to the first steroid binding domain-like region. The cytosolic domain is found to contain three helices, and the secondary structure and backbone dynamics of the chaperone domain are consistent with that determined previously for the chaperone domain alone. The position of TM2 provides a framework for ongoing studies of S1R ligand binding and oligomerisation.

17 páginas, 8 figuras, 1 tabla. Este fichero contiene 5 figuras y 3 tablas de material suplementario, The CD2AP (CD2-associated protein) and CIN85 (Cbl-interacting protein of 85 kDa) adaptor proteins each employ three Src homology 3 (SH3) domains to cluster protein partners and ensure efficient signal transduction and down-regulation of tyrosine kinase receptors. Using NMR, isothermal titration calorimetry and small-angle X-ray scattering methods, we have characterized several binding modes of the N-terminal SH3 domain (SH3A) of CD2AP and CIN85 with two natural atypical proline-rich regions in CD2 (cluster of differentiation 2) and Cbl-b (Casitas B-lineage lymphoma), and compared these data with previous studies and published crystal structures. Our experiments show that the CD2AP-SH3A domain forms a type II dimer with CD2 and both type I and type II dimeric complexes with Cbl-b. Like CD2AP, the CIN85-SH3A domain forms a type II complex with CD2, but a trimeric complex with Cbl-b, whereby the type I and II interactions take place at the same time. Together, these results explain how multiple interactions among similar SH3 domains and ligands produce a high degree of diversity in tyrosine kinase, cell adhesion or T-cell signaling pathways., This research is funded by grant FQM02838 from the Junta de Andalucía. M.A.C. is supported by an FPU grant from the Spanish Ministry of Education (MEC). A.G-P is an FWO post-doc fellow. J.L.OR is a FEBS post-doc fellow. Work in NvN laboratory is supported by the VIB and the Flanders Hercules Foundation. Work in JB laboratory was supported by Ministerio de Ciencia e Innovación (SAF2009-10667, SAF2012-31405) and Generalitat Valenciana ACOMP/2012/024.

The precision with which X-ray crystallography and nuclear magnetic resonance (NMR) have provided structural models of biologically active and inactive conformations of countless proteins belies an easily overlooked dilemma. Proteins are inherently dynamic, exhibiting conformational freedom on timescales from picoseconds to seconds, implicating structural rearrangements that are essential for their biological function. Classical structural biology determines static models, that afford little insight into the underlying conformational equilibrium. The role that structural dynamics play in biological processes can only be understood by characterizing all thermally accessible protein conformations and their populations. NMR spectroscopy is uniquely sensitive to the presence of conformational dynamics in solution. Residual dipolar couplings (RDCs) measured in weakly aligned proteins, scalar couplings, and chemical shifts, probe motions occurring on timescales faster than 100 s of microseconds. These parameters therefore offer general tools to characterize protein motion on physiologically important timescales. A common approach to the dynamic interpretation of RDCs is to combine experimental restraint terms with a classical potential-energy force field to develop a conformational ensemble in agreement with experimental data. RDCs have also been exploited to characterize the conformational space sampled by the protein backbone either by fitting experimental data to determine angular excursions of internuclear bond vectors, or in comparison with different levels of accelerated molecular dynamics (AMD) to describe the most appropriate ensemble. Comparison of motions modeled using the Gaussian axial fluctuation (GAF) model, with ensembles derived from restraint-free AMD, demonstrated that such methods can provide a convergent description of protein motion.

A complete understanding of the relationship between biological activity and molecular conformation requires an understanding of the thermally accessible potential energy landscape. An extensive set of experimental NMR residual dipolar couplings (RDCs) has been used to determine the conformational behavior of CD2AP SH3C on multiple timescales, using the Gaussian Axial Fluctuation model, and comparison to restraint-free accelerated molecular dynamics simulation. These robust analyses provide a comprehensive description of conformational fluctuations on picosecond to millisecond timescales. While the β-sheets show negligible slow motions, larger amplitude slow dynamics are found in the n-SRC and RT loops that mediate physiological interactions.

The description of the interactome represents one of key challenges remaining for structural biology. Physiologically important weak interactions, with dissociation constants above 100 μM, are remarkably common, but remain beyond the reach of most of structural biology. NMR spectroscopy, and in particular, residual dipolar couplings (RDCs) provide crucial conformational constraints on intermolecular orientation in molecular complexes, but the combination of free and bound contributions to the measured RDC seriously complicates their exploitation for weakly interacting partners. We develop a robust approach for the determination of weak complexes based on: (i) differential isotopic labeling of the partner proteins facilitating RDC measurement in both partners; (ii) measurement of RDC changes upon titration into different equilibrium mixtures of partially aligned free and complex forms of the proteins; (iii) novel analytical approaches to determine the effective alignment in all equilibrium mixtures; and (iv) extraction of precise RDCs for bound forms of both partner proteins. The approach is demonstrated for the determination of the three-dimensional structure of the weakly interacting CD2AP SH3-C:Ubiquitin complex (Kd = 132 ± 13 μM) and is shown, using cross-validation, to be highly precise. We expect this methodology to extend the remarkable and unique ability of NMR to study weak protein–protein complexes., Grant BIO2005-04650 from the Spanish Ministry of Education and Science (MEC); the Commisariat à l’Energie Atomique; the French Centre National pour la Recherche Scientifique; the Université Joseph Fourier, Grenoble; the French Research Ministry through ANR NT05-4_42781 (to M.B.); ANR JCJC05-0077 (to B.B.); a return grant of the Junta de Andalucia (to A.I.A.); Lundbeckfonden and a long-term EMBO fellowship (to M.R.J.). J.L.O.R. and N.A.J.v.N. are recipients of an FPU and Ramón y Cajal research contract from the MEC, respectively; and the Access to Research Infrastructures activity in the 6th Framework Programme of the EC (Contract # RII3-026145, EU-NMR). Funding for open access charge: ANR NT05-4_42781.