Search

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

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.

Regulation of the c-Abl (ABL1) tyrosine kinase is important because of its role in cellular signaling, and its relevance in the leukemiogenic counterpart (BCR-ABL). Both auto-inhibition and full activation of c-Abl are regulated by the interaction of the catalytic domain with the Src Homology 2 (SH2) domain. The mechanism by which this interaction enhances catalysis is not known. We combined computational simulations with mutagenesis and functional analysis to find that the SH2 domain conveys both local and global effects on the dynamics of the catalytic domain. Locally, it regulates the flexibility of the αC helix in a fashion reminiscent of cyclins in cyclin-dependent kinases, reorienting catalytically important motifs. At a more global level, SH2 binding redirects the hinge motion of the N and C lobes and changes the conformational equilibrium of the activation loop. The complex network of subtle structural shifts that link the SH2 domain with the activation loop and the active site may be partially conserved with other SH2-domain containing kinases and therefore offer additional parameters for the design of conformation-specific inhibitors., Author Summary The Abl kinase is a key player in many crucial cellular processes. It is also an important anti-cancer drug target, because a mutation leading to the fusion protein Bcr-Abl is the main cause for chronic myeloid leukemia (CML). Abl inhibitors are currently the only pharmaceutical treatment for CML. There are two main difficulties associated with the development of kinase inhibitors: the high similarity between active sites of different kinases, which makes selectivity a challenge, and mutations leading to resistance, which make it mandatory to search for alternative drugs. One important factor controlling Abl is the interplay between the catalytic domain and an SH2 domain. We used computer simulations to understand how the interactions between the domains modify the dynamic of the kinase and detected both local and global effects. Based on our computer model, we suggested mutations that should alter the domain-domain interplay. Consequently, we tested the mutants experimentally and found that they support our hypothesis. We propose that our findings can be of help for the development of new classes of Abl inhibitors, which would modify the domain-domain interplay instead of interfering directly with the active site.

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.

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.]