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PURPOSE: MDH2 (malate dehydrogenase 2) has recently been proposed as a novel potential pheochromocytoma/paraganglioma (PPGL) susceptibility gene, but its role in the disease has not been addressed. This study aimed to determine the prevalence of MDH2 pathogenic variants among PPGL patients and determine the associated phenotype. METHODS: Eight hundred thirty patients with PPGLs, negative for the main PPGL driver genes, were included in the study. Interpretation of variants of unknown significance (VUS) was performed using an algorithm based on 20 computational predictions, by implementing cell-based enzymatic and immunofluorescence assays, and/or by using a molecular dynamics simulation approach. RESULTS: Five variants with potential involvement in pathogenicity were identified: three missense (p.Arg104Gly, p.Val160Met and p.Ala256Thr), one in-frame deletion (p.Lys314del), and a splice-site variant (c.429+1G>T). All were germline and those with available biochemical data, corresponded to noradrenergic PPGL. CONCLUSION: This study suggests that MDH2 pathogenic variants may play a role in PPGL susceptibility and that they might be responsible for less than 1% of PPGLs in patients without pathogenic variants in other major PPGL driver genes, a prevalence similar to the one recently described for other PPGL genes. However, more epidemiological data are needed to recommend MDH2 testing in patients negative for other major PPGL genes.

SH2-containing-inositol-5-phosphatases (SHIPs) dephosphorylate the 5-phosphate of phosphatidylinositol-3,4,5-trisphosphate (PI(3,4,5)P3) and play important roles in regulating the PI3K/Akt pathway in physiology and disease. Aiming to uncover interdomain regulatory mechanisms in SHIP2, we determined crystal structures containing the 5-phosphatase and a proximal region adopting a C2 fold. This reveals an extensive interface between the two domains, which results in significant structural changes in the phosphatase domain. Both the phosphatase and C2 domains bind phosphatidylserine lipids, which likely helps to position the active site towards its substrate. Although located distant to the active site, the C2 domain greatly enhances catalytic turnover. Employing molecular dynamics, mutagenesis and cell biology, we identify two distinct allosteric signaling pathways, emanating from hydrophobic or polar interdomain interactions, differentially affecting lipid chain or headgroup moieties of PI(3,4,5)P3. Together, this study reveals details of multilayered C2-mediated effects important for SHIP2 activity and points towards interesting new possibilities for therapeutic interventions. We thank Jose´ Terro´ n Bautista for help with MD analysis. We thank the ESRF and ALBA for provid- ing the synchrotron-radiation facilities and the staff for their assistance in the data collection. We are grateful to the Barcelona Supercomputing Centre and National Supercomputing Centre (BSC-CNS) for allocating computer time to run the reported simulations. The work was supported by the Span- ish Ministry of Economy, Industry and Competitiveness (MEIC) Grants BFU2010-15923 (DL) and MEIC Project Retos BFU2016-77665-R co-funded by the European Regional Development Fund (ERDF) (DL), the Comunidad Auto´ noma de Madrid Grant S2010/BMD-2457 (DL), and by the National Cancer Research Centre. DL is also a recipient of awards from the Volkswagen Foundation (Az: 86 416–1) and Worldwide Cancer Research (15-1177). Sí

Alpha-solenoid proteins are suggested to constitute highly flexible macromolecules, whose structural variability and large surface area is instrumental in many important protein-protein binding processes. By equilibrium and nonequilibrium molecular dynamics simulations, we show that importin-beta, an archetypical alpha-solenoid, displays unprecedentedly large and fully reversible elasticity. Our stretching molecular dynamics simulations reveal full elasticity over up to twofold end-to-end extensions compared to its bound state. Despite the absence of any long-range intramolecular contacts, the protein can return to its equilibrium structure to within 3 A backbone RMSD after the release of mechanical stress. We find that this extreme degree of flexibility is based on an unusually flexible hydrophobic core that differs substantially from that of structurally similar but more rigid globular proteins. In that respect, the core of importin-beta resembles molten globules. The elastic behavior is dominated by nonpolar interactions between HEAT repeats, combined with conformational entropic effects. Our results suggest that alpha-solenoid structures such as importin-beta may bridge the molecular gap between completely structured and intrinsically disordered proteins.

Contains fulltext : 88268.pdf (Publisher’s version ) (Closed access) Transport of large proteins into the nucleus involves two events, binding of the cargo protein to a transport receptor in the cytoplasm and passage of the cargo-transporter complex through the selective permeability barrier of the nuclear pore complex. The permeability barrier is formed by largely disordered polypeptides, each containing a number of conserved hydrophobic phenylalanine-glycine (FG) sequence motifs, connected by hydrophilic linkers of varying sequence (FG nups). How the motifs interact to form the permeability barrier, however, is not yet known. We have, therefore, carried out molecular dynamics simulations on various model FG repeat peptides to study the aggregation propensity of FG nups and the specific roles of the hydrophobic FG motifs and the hydrophilic linkers. Our simulations show spontaneous aggregation of the model nups into hydrated aggregates, which exhibit structural features assumed to be part of the permeability barrier. Our simulations suggest that short beta-sheets are an important structural feature of the aggregates and that Phe residues are sufficiently exposed to allow rapid binding of transport receptors. A surprisingly large influence of the amino acid composition of the hydrophilic linkers on aggregation is seen, as well as a major contribution of hydrogen-bonding patterns.

In order to study the influence of Ser and Thr on the structure of transmembrane helices we have analyzed a database of helix stretches extracted from crystal structures of membrane proteins and an ensemble of model helices generated by molecular dynamics simulations. Both complementary analyses show that Ser and Thr in the g conformation induce and/or stabilize a structural distortion in the helix backbone. Using quantum mechanical calculations, we have attributed this effect to the electrostatic repulsion between the side chain Oc atom of Ser and Thr and the backbone carbonyl oxygen at position i 3. In order to minimize the repulsive force between these negatively charged oxygens, there is a modest increase of the helix bend angle as well as a local opening of the helix turn preceding Ser/Thr. This small distortion can be amplified through the helix, resulting in a significant displacement of the residues located at the other side of the helix. The crystal structures of aquaporin Z and the b2-adrenergic receptor are used to illustrate these effects. Ser/Thr-induced structural distortions can be implicated in processes as diverse as ligand recognition, protein function and protein folding.

To study the importance of charge–charge and cation-π interactions for the binding of positively charged amine ligands to their receptors, the energies of interaction between [(CH3)4–N]+, [(CH3)3–NH]+, and [(CH3)4–NH3]+ and acetate, as a model of Asp and Glu, and with benzene, as a model of aromatic side chains, were obtained at the MP2/aug-cc-pVDZ level of theory. The free energies of solvation in water were also calculated for the different amines. It was found that, although primary amines form stronger charge–charge interactions with acetate than tertiary or quaternary amines, the difference is not large enough to compensate their higher solvation energy. Quaternary amines show the weakest interaction with acetate. However, their alkyl groups can interact with various aromatic groups, enhancing ligand binding to the receptor. The analysis was completed with MD calculations on amine binding to the G protein-coupled receptors β2AR and CCR5. The calculations on the model systems were found to be in good agreement with the simulations of the ligand-receptor complexes.

The nuclear pore complex mediates nucleocytoplasmic transport of macromolecules in eukaryotic cells. Transport through the pore is restricted by a hydrophobic selectivity filter comprising disordered phenylalanine-glycine-rich repeats of nuclear pore proteins. Exchange through the pore requires specialized transport receptors, called exportins and importins, that interact with cargo proteins in a RanGTP-dependent manner. These receptors are highly flexible superhelical structures composed of HEAT-repeat motifs that adopt various degrees of extension in crystal structures. Here, we performed molecular-dynamics simulations using crystal structures of Importin-β in its free form or in complex with nuclear localization signal peptides as the starting conformation. Our simulations predicted that initially compact structures would adopt extended conformations in hydrophilic buffers, while contracted conformations would dominate in more hydrophobic solutions, mimicking the environment of the nuclear pore. We confirmed this experimentally by Förster resonance energy transfer experiments using dual-fluorophore-labeled Importin-β. These observations explain seemingly contradictory crystal structures and suggest a possible mechanism for cargo protection during passage of the nuclear pore. Such hydrophobic switching may be a general principle for environmental control of protein function.

The bond dissociation energies of the Co-C bonds in the cobalamin cofactors methylcobalamin and adenosylcobalamin were calculated using the hybrid quantum mechanics/molecular mechanics method IMOMM (integrated molecular orbital and molecular mechanics). Calculations were performed on models of differing complexities as well as on the full systems. We investigated the origin of the different experimental values for the Co-C bond dissociation energies in methylcobalamin and adenosylcobalamin, and have provided an explanation for the difficulties encountered when we attempt to reproduce this difference in quantum chemistry. Additional calculations have been performed using the Miertus-Scrocco-Tomasi method in order to estimate the influence of solvent effects on the homolytic Co-C bond cleavage. Introduction of these solvation effects is shown to be necessary for the correct reproduction of experimental trends in bond dissociation energies in solution, which consequently have no direct correlation with dissociation processes in the enzyme.

Density functional theory B3LYP calculations have been carried out on a large model for coenzyme B12. The dissociation curve for the homolytic cleavage of the Co–C bond has been studied for the free cofactor, in the gas phase and including the effect of a dielectric continuum with a dielectric constant of 4.00, as well as in the presence of some key residues present in methylmalonyl-CoA mutase, an enzyme that depends on coenzyme B12. We describe a hitherto unknown effect of the cofactor which facilitates the homolysis reaction: electrostatic interactions stabilize the radicals formed in this step. H-bonding to protein residues leads to further stabilization.

Density functional theory (DFT) Becke3LYP calculations including full and restricted geometry optimizations are carried out on the complex [Co(Cor)(Benz)(CH 3 )] + (Cor = corrin, Benz = benzimidazole), which is a model of B 1 2 cofactors, and on the products of the two possible heterolytic cleavages of the Co-C bond, [Co(Cor)(Benz)(CH 3 )], CH 3 +, [Co(Cor)(Benz)(CH 3 )] 2 + , and CH 3 -. The thermodynamics of the heterolytic processes are found to depend very significantly on the distance of the axial ligand from the cobalt. The results are explained through simple molecular orbital reasonings, and their possible implications for the biological reactivity of adenosylcobalamin and methylcobalamin are discussed.

Density functional theory (DFT) Becke3LYP calculations including full and restricted geometry optimizations are carried out on the complexes [Co(Cor)(Benz)(CH3)] (Cor = corrin, Benz = benzimidazole), [Co(Cor)(Benz)], [Co(Cor)(CH3)], and [Co(Cor)]. These systems, despite the absence of side-chains, constitute the most realistic models used to date for DFT calculations on cofactor B12 and its homolysis product. The calculations prove that both thermodynamics and kinetics of the homolytic bond cleavage of the Co−C bond have very little dependence on the position of the axial benzimidazole ligand. The generality of these results is confirmed by additional calculations on [Co(Cor)(Benz)(CH2R)] (R = tetrahydrofuran), [Co(Cor)(Im)(CH3)] (Im = imidazole), and [Co(Cor−CH3)(Benz)(CH3)] (Cor−CH3 = methylated corrin).

[RuH2Cl2(PiPr3)2] reacts with terminal alkynes to give the vinylidene complex [RuCl2(CCHR)(PiPr3)2]. As a side product the carbene complex [RuCl2(CHR)(PiPr3)2] is formed. The formation of the vinylidene compound has been studied widely and is well understood whereas the reaction mechanism that leads to the carbene complex is still unclear. We have studied two possible reaction paths at the B3LYP level of theory: on the one hand the addition of acetylene and two subsequent 1,3-H shifts from the metal center to the C2 carbon of the acetylene ligand; on the other hand the dissociation of HCl from the starting compound, rearrangement of acetylene to vinyl and the formation of the carbene by addition of HCl. Both reaction paths have been found to be possible. The former can be understood as a 1,3-H shift followed by a 1,2-H shift due to the unusual η2 coordination mode of the vinyl intermediate. The latter proceeds via protonation of the vinyl ligand and addition of Cl− to the metal center.

Experimental and computational dynamic force spectroscopy is widely used to determine the mechanical properties of single biomolecules. Whereas so far the focus has mainly been on rupture or unfolding forces, recent force-probe molecular dynamics simulations have revealed a strong loading rate dependence of biomolecular elasticities, which cannot be explained by the established one-dimensional transition-state treatments. We show that this nonequilibrium behavior can be explained by a theory that includes relaxation effects. For three structurally and mechanically quite diverse systems, a single relaxation mode suffices to quantitatively describe their loading-rate-dependent elastic behavior. Atomistic simulations of these systems revealed the microscopic nature of the respective relaxation modes. This result suggests a new type of ‘‘elasticity spectroscopy’’ experiment, which should render nonequilibrium properties of structured macromolecules accessible to single-molecule force spectroscopy.

c-Src and c-Abl are two closely related protein kinases that constitute important anticancer targets. Despite their high sequence identity, they show different sensitivities to the anticancer drug imatinib, which binds specifically to a particular inactive conformation in which the Asp of the conserved DFG motif points outward (DFG-out). We have analyzed the DFG conformational transition of the two kinases using massive molecular dynamics simulations, free energy calculations, and isothermal titration calorimetry. On the basis of the reconstruction of the free energy surfaces for the DFG-in to DFG-out conformational changes of c-Src and c-Abl, we propose that the different flexibility of the two kinases results in a different stability of the DFG-out conformation and might be the main determinant of imatinib selectivity.

G protein-coupled receptors (GPCRs) interact with an extraordinary diversity of ligands by means of their extracellular domains and/or the extracellular part of the transmembrane (TM) segments. Each receptor subfamily has developed specific sequence motifs to adjust the structural characteristics of its cognate ligands to a common set of conformational rearrangements of the TM segments near the G protein binding domains during the activation process. Thus, GPCRs have fulfilled this adaptation during their evolution by customizing a preserved 7TM scaffold through conformational plasticity. We use this term to describe the structural differences near the binding site crevices among different receptor subfamilies, responsible for the selective recognition of diverse ligands among different receptor subfamilies. By comparing the sequence of rhodopsin at specific key regions of the TM bundle with the sequences of other GPCRs we have found that the extracellular region of TMs 2 and 3 provides a remarkable example of conformational plasticity within Class A GPCRs. Thus, rhodopsin-based molecular models need to include the plasticity of the binding sites among GPCR families, since the "quality" of these homology models is intimately linked with the success in the processes of rational drug-design or virtual screening of chemical databases.

Single molecule experiments such as atomic force microscopy, optical tweezers or force probe molecular dynamics have been used extensively to determine mechanical properties of biological macromolecules. In most cases, yielding or unbinding forces were found to depend on the loading rate according to Bell's model. The one-dimensional model predicts a logarithmic dependence of yielding forces on the loading rate, but velocity-independent elastic constants. Also more recent, refined one-dimensional treatments share this property. However, in recent molecular dynamics simulations of the nuclear transport receptor importin-beta and the outer shell of the southern bean mosaic virus, strongly velocity dependent elastic constants were seen, with yielding forces following Bell's model.To resolve this discrepancy, we here present a two-dimensional relaxation model and show that it can explain all elastic properties seen in the atomistic simulations. In our model, relaxation modes perpendicular to the reaction coordinate are allowed to fluctuate and relax independently of the reaction coordinate. Contrary to Bell's model, a stiffening of macromolecules at high probe velocities can be explained, and the corresponding forces are correctly predicted. Combined with simple rate theory, our model also predicts the observed logarithmic dependence of yielding forces on the probe velocity.

The application of quantum mechanics/molecular mechanics (QM/MM) methods in transition metal chemistry is growing steadily. It becomes therefore appropriate to assess the importance of a number of technical issues associated to their implementation. This work presents the discussion of several of these issues, including the eventual need for conformational searches, the choice of the MM force field and the possibility of its tuning. The examples presented here prove that a proper handling of these technical aspects can lead to an improvement in the efficiency and quality of QM/MM calculations.

Structure 21(8), 1350 - 1360 (2013). doi:10.1016/j.str.2013.05.015, Proteins carrying nuclear export signals cooperatively assemble with the export factor CRM1 and the effector protein RanGTP. In lower eukaryotes, this cooperativity is coupled to CRM1 conformational changes; however, it is unknown if mammalian CRM1 maintains its compact conformation or shows similar structural flexibility. Here, combinations of small-angle X-ray solution scattering and electron microscopy experiments with molecular dynamics simulations reveal pronounced conformational flexibility in mammalian CRM1 and demonstrate that RanGTP binding induces association of its N- and C-terminal regions to form a toroid structure. The CRM1 toroid is stabilized mainly by local interactions between the terminal regions, rather than by global strain. The CRM1 acidic loop is key in transmitting the effect of this RanGTP-induced global conformational change to the NES-binding cleft by shifting its population to the open state, which displays enhanced cargo affinity. Cooperative CRM1 export complex assembly thus constitutes a highly dynamic process, encompassing an intricate interplay of global and local structural changes., Published by Elsevier Science, London [u.a.]

In eukaryotic cells, exchange of macromolecules between the cytoplasm and the nucleus is mediated by specialized transport factors. By binding to these transporters, cargo molecules which are otherwise excluded from entering the nucleus can traverse the nuclear pore efficiently. Most of the proteins mediating nuclear import and export belong to the importin beta family. The transport cycle of importin beta starts with recognition of the cargo in the cytoplasm. The importin-cargo complex then crosses the permeability barrier of the nuclear pore and enters the nucleus, where the complex dissociates upon binding of RanGTP to importin beta.Importin beta has a superhelical structure and exhibits a great intrinsic flexibility, which is needed for recognition of a wide variety of ligands [1]. Although small angle scattering data, as well as previous molecular dynamics studies, indicate that in solution importin beta adopts a rather open conformation, most crystal structures exhibit a compact conformation of the superhelix [2, 3].We carried out all-atom molecular dynamics simulations of importin beta in aqueous solution and in hydrophobic media and found a large influence of the hydrophobicity of the environment on conformation and dynamical properties of importin beta. Our results indicate that the structural plasticity of importin beta is not only important for cargo recognition, but that its dependence on the environment plays an important role in the transport process. We propose a mechanism by which opening and closing of importin beta during nuclear import facilitate cargo binding, transport and release.[1] Conti, E.; Muller, C. W. & Stewart, M. Curr Opin Struct Biol 2006, 16, 237-244.[2] Fukuhara, N.; Fernandez, E.; Ebert, J.; Conti, E. & Svergun, D. J Biol Chem 2004, 279, 2176-2181.[3] Zachariae, U. & Grubmuller, H. Structure 2008, 16, 906-915.