SH2-containing-inositol-5-phosphatases (SHIPs) dephosphorylate the 5-phosphate of phosphatidylinositol-3,4,5-trisphosphate (PI(3,4,5)P
) 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)P
. Together, this study reveals details of multilayered C2-mediated effects important for SHIP2 activity and points towards interesting new possibilities for therapeutic interventions.
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.
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.
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.
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.
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.
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-β, an archetypical α-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 Å 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-β resembles molten globules. The elastic behavior is dominated by nonpolar interactions between HEAT repeats, combined with conformational entropic effects. Our results suggest that α-solenoid structures such as importin-β may bridge the molecular gap between completely structured and intrinsically disordered proteins.
Not so watered down. In this review, we summarize the role of water molecules embedded in the transmembrane bundle of G protein‐coupled receptors in stabilizing intra‐ and interhelical interactions, ...and their use for building computer‐generated homology models.
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.
•Hybrid methods show mammalian CRM1 as highly flexible•Free human CRM1 exists in extended superhelical and compact toroidal states•RanGTP binding to CRM1 reduces CRM flexibility by shifting it to compact forms•Acidic loop modulates the open-to-closed nuclear export signal cargo-binding cleft
Using hybrid methods, Dölker et al. reveal the high flexibility of mammalian exportin CRM1 behind an intriguing coupling between overall conformation and local positional changes. The rearrangements mediate cooperativity between the RanGTP- and the cargo-binding sites, resulting in remarkable long distance coupling.
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 Oγ 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 β
2-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.
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.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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 (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).
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