ResearchPad - salt-bridges https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[Crystal structure of <i>Thermus thermophilus</i> methylenetetrahydrofolate dehydrogenase and determinants of thermostability]]> https://www.researchpad.co/article/elastic_article_13865 The elucidation of mechanisms behind the thermostability of proteins is extremely important both from the theoretical and applied perspective. Here we report the crystal structure of methylenetetrahydrofolate dehydrogenase (MTHFD) from Thermus thermophilus HB8, a thermophilic model organism. Molecular dynamics trajectory analysis of this protein at different temperatures (303 K, 333 K and 363 K) was compared with homologous proteins from the less temperature resistant organism Thermoplasma acidophilum and the mesophilic organism Acinetobacter baumannii using several data reduction techniques like principal component analysis (PCA), residue interaction network (RIN) analysis and rotamer analysis. These methods enabled the determination of important residues for the thermostability of this enzyme. The description of rotamer distributions by Gini coefficients and Kullback–Leibler (KL) divergence both revealed significant correlations with temperature. The emerging view seems to indicate that a static salt bridge/charged residue network plays a fundamental role in the temperature resistance of Thermus thermophilus MTHFD by enhancing both electrostatic interactions and entropic energy dispersion. Furthermore, this analysis uncovered a relationship between residue mutations and evolutionary pressure acting on thermophilic organisms and thus could be of use for the design of future thermostable enzymes.

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<![CDATA[CRISPR-Cas9 In Situ engineering of subtilisin E in Bacillus subtilis]]> https://www.researchpad.co/article/5c3d0133d5eed0c48403922d

CRISPR-Cas systems have become widely used across all fields of biology as a genome engineering tool. With its recent demonstration in the Gram positive industrial workhorse Bacillus subtilis, this tool has become an attractive option for rapid, markerless strain engineering of industrial production hosts. Previously described strategies for CRISPR-Cas9 genome editing in B. subtilis have involved chromosomal integrations of Cas9 and single guide RNA expression cassettes, or construction of large plasmids for simultaneous transformation of both single guide RNA and donor DNA. Here we use a flexible, co-transformation approach where the single guide RNA is inserted in a plasmid for Cas9 co-expression, and the donor DNA is supplied as a linear PCR product observing an editing efficiency of 76%. This allowed multiple, rapid rounds of in situ editing of the subtilisin E gene to incorporate a salt bridge triad present in the Bacillus clausii thermotolerant homolog, M-protease. A novel subtilisin E variant was obtained with increased thermotolerance and activity.

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<![CDATA[Structural Insights into Streptococcal Competence Regulation by the Cell-to-Cell Communication System ComRS]]> https://www.researchpad.co/article/5989daa8ab0ee8fa60ba821f

In Gram-positive bacteria, cell-to-cell communication mainly relies on extracellular signaling peptides, which elicit a response either indirectly, by triggering a two-component phosphorelay, or directly, by binding to cytoplasmic effectors. The latter comprise the RNPP family (Rgg and original regulators Rap, NprR, PrgX and PlcR), whose members regulate important bacterial processes such as sporulation, conjugation, and virulence. RNPP proteins are increasingly considered as interesting targets for the development of new antibacterial agents. These proteins are characterized by a TPR-type peptide-binding domain, and except for Rap proteins, also contain an N-terminal HTH-type DNA-binding domain and display a transcriptional activity. Here, we elucidate the structure-function relationship of the transcription factor ComR, a new member of the RNPP family, which positively controls competence for natural DNA transformation in streptococci. ComR is directly activated by the binding of its associated pheromone XIP, the mature form of the comX/sigX-inducing-peptide ComS. The crystal structure analysis of ComR from Streptococcus thermophilus combined with a mutational analysis and in vivo assays allows us to propose an original molecular mechanism of the ComR regulation mode. XIP-binding induces release of the sequestered HTH domain and ComR dimerization to allow DNA binding. Importantly, we bring evidence that this activation mechanism is conserved and specific to ComR orthologues, demonstrating that ComR is not an Rgg protein as initially proposed, but instead constitutes a new member of the RNPP family. In addition, identification of XIP and ComR residues important for competence activation constitutes a crucial step towards the design of antagonistic strategies to control gene exchanges among streptococci.

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<![CDATA[Acetyl group coordinated progression through the catalytic cycle of an arylalkylamine N-acetyltransferase]]> https://www.researchpad.co/article/5989db5aab0ee8fa60bdf65c

The transfer of an acetyl group from acetyl-CoA to an acceptor amine is a ubiquitous biochemical transformation catalyzed by Gcn5-related N-acetyltransferases (GNATs). Although it is established that the reaction proceeds through a sequential ordered mechanism, the role of the acetyl group in driving the ordered formation of binary and ternary complexes remains elusive. Herein, we show that CoA and acetyl-CoA alter the conformation of the substrate binding site of an arylalkylamine N-acetyltransferase (AANAT) to facilitate interaction with acceptor substrates. However, it is the presence of the acetyl group within the catalytic funnel that triggers high affinity binding. Acetyl group occupancy is relayed through a conserved salt bridge between the P-loop and the acceptor binding site, and is manifested as differential dynamics in the CoA and acetyl-CoA-bound states. The capacity of the acetyl group carried by an acceptor to promote its tight binding even in the absence of CoA, but also its mutually exclusive position to the acetyl group of acetyl-CoA underscore its importance in coordinating the progression of the catalytic cycle.

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<![CDATA[The Dynamics of the Human Leukocyte Antigen Head Domain Modulates Its Recognition by the T-Cell Receptor]]> https://www.researchpad.co/article/5989d9feab0ee8fa60b73113

Generating the immune response requires the discrimination of peptides presented by the human leukocyte antigen complex (HLA) through the T-cell receptor (TCR). However, how a single amino acid substitution in the antigen bonded to HLA affects the response of T cells remains uncertain. Hence, we used molecular dynamics computations to analyze the molecular interactions between peptides, HLA and TCR. We compared immunologically reactive complexes with non-reactive and weakly reactive complexes. MD trajectories were produced to simulate the behavior of isolated components of the various p-HLA-TCR complexes. Analysis of the fluctuations showed that p-HLA binding barely restrains TCR motions, and mainly affects the CDR3 loops. Conversely, inactive p-HLA complexes displayed significant drop in their dynamics when compared with its free versus ternary forms (p-HLA-TCR). In agreement, the free non-reactive p-HLA complexes showed a lower amount of salt bridges than the responsive ones. This resulted in differences between the electrostatic potentials of reactive and inactive p-HLA species and larger vibrational entropies in non-elicitor complexes. Analysis of the ternary p-HLA-TCR complexes also revealed a larger number of salt bridges in the responsive complexes. To summarize, our computations indicate that the affinity of each p-HLA complex towards TCR is intimately linked to both, the dynamics of its free species and its ability to form specific intermolecular salt-bridges in the ternary complexes. Of outstanding interest is the emerging concept of antigen reactivity involving its interplay with the HLA head sidechain dynamics by rearranging its salt-bridges.

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<![CDATA[The Functional Unit of Neisseria meningitidis 3-Deoxy-ᴅ-Arabino-Heptulosonate 7-Phosphate Synthase Is Dimeric]]> https://www.researchpad.co/article/5989db39ab0ee8fa60bd452a

Neisseria meningitidis 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (NmeDAH7PS) adopts a homotetrameric structure consisting of an extensive and a less extensive interface. Perturbation of the less extensive interface through a single mutation of a salt bridge (Arg126-Glu27) formed at the tetramer interface of all chains resulted in a dimeric DAH7PS in solution, as determined by small angle X-ray scattering, analytical ultracentrifugation and analytical size-exclusion chromatography. The dimeric NmeDAH7PSR126S variant was shown to be catalytically active in the aldol-like condensation reaction between d-erythrose 4-phosphate and phosphoenolpyruvate, and allosterically inhibited by l-phenylalanine to the same extent as the wild-type enzyme. The dimeric NmeDAH7PSR126S variant exhibited a slight reduction in thermal stability by differential scanning calorimetry experiments and a slow loss of activity over time compared to the wild-type enzyme. Although NmeDAH7PSR126S crystallised as a tetramer, like the wild-type enzyme, structural asymmetry at the less extensive interface was observed consistent with its destabilisation. The tetrameric association enabled by this Arg126-Glu27 salt-bridge appears to contribute solely to the stability of the protein, ultimately revealing that the functional unit of NmeDAH7PS is dimeric.

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<![CDATA[Effect of Glu12-His89 Interaction on Dynamic Structures in HIV-1 p17 Matrix Protein Elucidated by NMR]]> https://www.researchpad.co/article/5989dac2ab0ee8fa60bb0f77

To test the existence of the salt bridge and stability of the HIV-1 p17 matrix protein, an E12A (mutated at helix 1) was established to abolish possible electrostatic interactions. The chemical shift perturbation from the comparison between wild type and E12A suggested the existence of an electrostatic interaction in wild type between E12 and H89 (located in helix 4). Unexpectedly, the studies using urea denaturation indicated that the E12A substitution slightly stabilized the protein. The dynamic structure of E12A was examined under physiological conditions by both amide proton exchange and relaxation studies. The quick exchange method of amide protons revealed that the residues with faster exchange were located at the mutated region, around A12, compared to those of the wild-type protein. In addition, some residues at the region of helix 4, including H89, exhibited faster exchange in the mutant. In contrast, the average values of the kinetic rate constants for amide proton exchange for residues located in all loop regions were slightly lower in E12A than in wild type. Furthermore, the analyses of the order parameter revealed that less flexible structures existed at each loop region in E12A. Interestingly, the structures of the regions including the alpha1-2 loop and helix 5 of E12A exhibited more significant conformational exchanges with the NMR time-scale than those of wild type. Under lower pH conditions, for further destabilization, the helix 1 and alpha2-3 loop in E12A became more fluctuating than at physiological pH. Because the E12A mutant lacks the activities for trimer formation on the basis of the analytical ultra-centrifuge studies on the sedimentation distribution of p17 (Fledderman et al. Biochemistry 49, 9551–9562, 2010), it is possible that the changes in the dynamic structures induced by the absence of the E12-H89 interaction in the p17 matrix protein contributes to a loss of virus assembly.

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<![CDATA[Molecular Dynamics Simulations and Classical Multidimensional Scaling Unveil New Metastable States in the Conformational Landscape of CDK2]]> https://www.researchpad.co/article/5989da5aab0ee8fa60b8fd1e

Protein kinases are key regulatory nodes in cellular networks and their function has been shown to be intimately coupled with their structural flexibility. However, understanding the key structural mechanisms of large conformational transitions remains a difficult task. CDK2 is a crucial regulator of cell cycle. Its activity is finely tuned by Cyclin E/A and the catalytic segment phosphorylation, whereas its deregulation occurs in many types of cancer. ATP competitive inhibitors have failed to be approved for clinical use due to toxicity issues raised by a lack of selectivity. However, in the last few years type III allosteric inhibitors have emerged as an alternative strategy to selectively modulate CDK2 activity. In this study we have investigated the conformational variability of CDK2. A low dimensional conformational landscape of CDK2 was modeled using classical multidimensional scaling on a set of 255 crystal structures. Microsecond-scale plain and accelerated MD simulations were used to populate this landscape by using an out-of-sample extension of multidimensional scaling. CDK2 was simulated in the apo-form and in complex with the allosteric inhibitor 8-anilino-1-napthalenesulfonic acid (ANS). The apo-CDK2 landscape analysis showed a conformational equilibrium between an Src-like inactive conformation and an active-like form. These two states are separated by different metastable states that share hybrid structural features with both forms of the kinase. In contrast, the CDK2/ANS complex landscape is compatible with a conformational selection picture where the binding of ANS in proximity of the αC helix causes a population shift toward the inactive conformation. Interestingly, the new metastable states could enlarge the pool of candidate structures for the development of selective allosteric CDK2 inhibitors. The method here presented should not be limited to the CDK2 case but could be used to systematically unmask similar mechanisms throughout the human kinome.

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<![CDATA[Crystal Structure of C-Terminal Coiled-Coil Domain of SYCP1 Reveals Non-Canonical Anti-Parallel Dimeric Structure of Transverse Filament at the Synaptonemal Complex]]> https://www.researchpad.co/article/5989da1eab0ee8fa60b7dfd4

The synaptonemal complex protein 1 (SYCP1) is the main structural element of transverse filaments (TFs) of the synaptonemal complex (SC), which is a meiosis-specific complex structure formed at the synapse of homologue chromosomes to hold them together. The N-terminal domain of SYCP1 is known to be located within the central elements (CEs), whereas the C-terminal domain is located toward lateral elements (LEs). SYCP1 is a well-known meiosis marker that is also known to be a prognostic marker in the early stage of several cancers including breast, gliomas, and ovarian cancers. The structure of SC, especially the TF structure formed mainly by SYCP1, remains unclear without any structural information. To elucidate a molecular basis of SC formation and function, we first solved the crystal structure of C-terminal coiled-coil domain of SYCP1. The coiled-coil domain of SYCP1 forms asymmetric, anti-parallel dimers in solution.

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<![CDATA[Dodecylphosphocholine Micelles Induce Amyloid Formation of the PrP(110-136) Peptide via an α-Helical Metastable Conformation]]> https://www.researchpad.co/article/5989d9ffab0ee8fa60b737e3

A peptide encompassing the conserved hydrophobic region and the first β-strand of the prion protein (PrP(110–136)) shown to interact with the surface of dodecylphosphocholine micelles adopts an α-helical conformation that is localized below the head-group layer. This surface-bound peptide has a half-life of one day, and readily initiates the formation of amyloid fibrils. The presence of the latter was confirmed using birefringence microscopy upon Congo red binding and thioflavin T-binding induced fluorescence. The observation of this metastable α-helical conformer provides a unique snapshot of the early steps of the inter-conversion pathway. These findings together with the body of evidence from the prion literature allowed us to propose a mechanism for the conversion of PrPC to amyloid material.

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<![CDATA[Is the Conformational Ensemble of Alzheimer’s Aβ10-40 Peptide Force Field Dependent?]]> https://www.researchpad.co/article/5989db54ab0ee8fa60bdd142

By applying REMD simulations we have performed comparative analysis of the conformational ensembles of amino-truncated Aβ10-40 peptide produced with five force fields, which combine four protein parameterizations (CHARMM36, CHARMM22*, CHARMM22/cmap, and OPLS-AA) and two water models (standard and modified TIP3P). Aβ10-40 conformations were analyzed by computing secondary structure, backbone fluctuations, tertiary interactions, and radius of gyration. We have also calculated Aβ10-40 3JHNHα-coupling and RDC constants and compared them with their experimental counterparts obtained for the full-length Aβ1-40 peptide. Our study led us to several conclusions. First, all force fields predict that Aβ adopts unfolded structure dominated by turn and random coil conformations. Second, specific TIP3P water model does not dramatically affect secondary or tertiary Aβ10-40 structure, albeit standard TIP3P model favors slightly more compact states. Third, although the secondary structures observed in CHARMM36 and CHARMM22/cmap simulations are qualitatively similar, their tertiary interactions show little consistency. Fourth, two force fields, OPLS-AA and CHARMM22* have unique features setting them apart from CHARMM36 or CHARMM22/cmap. OPLS-AA reveals moderate β-structure propensity coupled with extensive, but weak long-range tertiary interactions leading to Aβ collapsed conformations. CHARMM22* exhibits moderate helix propensity and generates multiple exceptionally stable long- and short-range interactions. Our investigation suggests that among all force fields CHARMM22* differs the most from CHARMM36. Fifth, the analysis of 3JHNHα-coupling and RDC constants based on CHARMM36 force field with standard TIP3P model led us to an unexpected finding that in silico Aβ10-40 and experimental Aβ1-40 constants are generally in better agreement than these quantities computed and measured for identical peptides, such as Aβ1-40 or Aβ1-42. This observation suggests that the differences in the conformational ensembles of Aβ10-40 and Aβ1-40 are small and the former can be used as proxy of the full-length peptide. Based on this argument, we concluded that CHARMM36 force field with standard TIP3P model produces the most accurate representation of Aβ10-40 conformational ensemble.

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<![CDATA[Decoding the Interactions Regulating the Active State Mechanics of Eukaryotic Protein Kinases]]> https://www.researchpad.co/article/5989daacab0ee8fa60ba9bf4

Eukaryotic protein kinases regulate most cellular functions by phosphorylating targeted protein substrates through a highly conserved catalytic core. In the active state, the catalytic core oscillates between open, intermediate, and closed conformations. Currently, the intramolecular interactions that regulate the active state mechanics are not well understood. Here, using cAMP-dependent protein kinase as a representative model coupled with biochemical, biophysical, and computational techniques, we define a set of highly conserved electrostatic and hydrophobic interactions working harmoniously to regulate these mechanics. These include the previously identified salt bridge between a lysine from the β3-strand and a glutamate from the αC-helix as well as an electrostatic interaction between the phosphorylated activation loop and αC-helix and an ensemble of hydrophobic residues of the Regulatory spine and Shell. Moreover, for over three decades it was thought that the highly conserved β3-lysine was essential for phosphoryl transfer, but our findings show that the β3-lysine is not required for phosphoryl transfer but is essential for the active state mechanics.

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<![CDATA[Allosteric Regulation in the Ligand Binding Domain of Retinoic Acid Receptorγ]]> https://www.researchpad.co/article/5989db4fab0ee8fa60bdb949

Retinoic acid (RA) plays key roles in cell differentiation and growth arrest through nuclear retinoic acid receptors (RARs), which are ligand-dependent transcription factors. While the main trigger of RAR activation is the binding of RA, phosphorylation of the receptors has also emerged as an important regulatory signal. Phosphorylation of the RARγ N-terminal domain (NTD) is known to play a functional role in neuronal differentiation. In this work, we investigated the phosphorylation of RARγ ligand binding domain (LBD), and present evidence that the phosphorylation status of the LBD affects the phosphorylation of the NTD region. We solved the X-ray structure of a phospho-mimetic mutant of the LBD (RARγ S371E), which we used in molecular dynamics simulations to characterize the consequences of the S371E mutation on the RARγ structural dynamics. Combined with simulations of the wild-type LBD, we show that the conformational equilibria of LBD salt bridges (notably R387-D340) are affected by the S371E mutation, which likely affects the recruitment of the kinase complex that phosphorylates the NTD. The molecular dynamics simulations also showed that a conservative mutation in this salt bridge (R387K) affects the dynamics of the LBD without inducing large conformational changes. Finally, cellular assays showed that the phosphorylation of the NTD of RARγ is differentially regulated by retinoic acid in RARγWT and in the S371N, S371E and R387K mutants. This multidisciplinary work highlights an allosteric coupling between phosphorylations of the LBD and the NTD of RARγ and supports the importance of structural dynamics involving electrostatic interactions in the regulation of RARs activity.

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<![CDATA[Viral Evolved Inhibition Mechanism of the RNA Dependent Protein Kinase PKR's Kinase Domain, a Structural Perspective]]> https://www.researchpad.co/article/5989d9d3ab0ee8fa60b64cdc

The protein kinase PKR activated by viral dsRNA, phosphorylates the eIF2α, which inhibit the mechanism of translation initiation. Viral evolved proteins mimicking the eIF2α block its phosphorylation and help in the viral replication. To decipher the molecular basis for the PKR’s substrate and inhibitor interaction mechanisms, we carried the molecular dynamics studies on the catalytic domain of PKR in complex with substrate eIF2α, and inhibitors TAT and K3L. The studies conducted show the altered domain movements of N lobe, which confers open and close state to the substrate-binding cavity. In addition, PKR exhibits variations in the secondary structural transition of the activation loop residues, and inter molecular contacts with the substrate and the inhibitors. Phosphorylation of the P+1 loop at the Thr-451 increases the affinity of the binding proteins exhibiting its role in the phosphorylation events. The implications of structural mechanisms uncovered will help to understand the basis of the evolution of the host-viral and the viral replication mechanisms.

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<![CDATA[SARAH Domain-Mediated MST2-RASSF Dimeric Interactions]]> https://www.researchpad.co/article/5989dab1ab0ee8fa60bab660

RASSF enzymes act as key apoptosis activators and tumor suppressors, being downregulated in many human cancers, although their exact regulatory roles remain unknown. A key downstream event in the RASSF pathway is the regulation of MST kinases, which are main effectors of RASSF-induced apoptosis. The regulation of MST1/2 includes both homo- and heterodimerization, mediated by helical SARAH domains, though the underlying molecular interaction mechanism is unclear. Here, we study the interactions between RASSF1A, RASSF5, and MST2 SARAH domains by using both atomistic molecular simulation techniques and experiments. We construct and study models of MST2 homodimers and MST2-RASSF SARAH heterodimers, and we identify the factors that control their high molecular stability. In addition, we also analyze both computationally and experimentally the interactions of MST2 SARAH domains with a series of synthetic peptides particularly designed to bind to it, and hope that our approach can be used to address some of the challenging problems in designing new anti-cancer drugs.

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<![CDATA[Structural and Functional Effect of an Oscillating Electric Field on the Dopamine-D3 Receptor: A Molecular Dynamics Simulation Study]]> https://www.researchpad.co/article/5989db28ab0ee8fa60bd0c18

Dopamine as a neurotransmitter plays a critical role in the functioning of the central nervous system. The structure of D3 receptor as a member of class A G-protein coupled receptors (GPCRs) has been reported. We used MD simulation to investigate the effect of an oscillating electric field, with frequencies in the range 0.6–800 GHz applied along the z-direction, on the dopamine-D3R complex. The simulations showed that at some frequencies, the application of an external oscillating electric field along the z-direction has a considerable effect on the dopamine-D3R. However, there is no enough evidence for prediction of changes in specific frequency, implying that there is no order in changes. Computing the correlation coefficient parameter showed that increasing the field frequency can weaken the interaction between dopamine and D3R and may decrease the Arg128{3.50}-Glu324{6.30} distance. Because of high stability of α helices along the z-direction, applying an oscillating electric field in this direction with an amplitude 10-time higher did not have a considerable effect. However, applying the oscillating field at the frequency of 0.6 GHz along other directions, such as X-Y and Y-Z planes, could change the energy between the dopamine and the D3R, and the number of internal hydrogen bonds of the protein. This can be due to the effect of the direction of the electric field vis-à-vis the ligands orientation and the interaction of the oscillating electric field with the dipole moment of the protein.

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<![CDATA[Bending-Twisting Motions and Main Interactions in Nucleoplasmin Nuclear Import]]> https://www.researchpad.co/article/5989da2bab0ee8fa60b8263e

Alpha solenoid proteins play a key role in regulating the classical nuclear import pathway, recognizing a target protein and transporting it into the nucleus. Importin-α (Impα) is the solenoid responsible for cargo protein recognition, and it has been extensively studied by X-ray crystallography to understand the binding specificity. To comprehend the main motions of Impα and to extend the information about the critical interactions during carrier-cargo recognition, we surveyed different conformational states based on molecular dynamics (MD) and normal mode (NM) analyses. Our model of study was a crystallographic structure of Impα complexed with the classical nuclear localization sequence (cNLS) from nucleoplasmin (Npl), which was submitted to multiple 100 ns of MD simulations. Representative conformations were selected for calculating the 87 lowest frequencies NMs of vibration, and a displacement approach was applied along each NM. Based on geometric criteria, using the radius of curvature and inter-repeat angles as the reference metrics, the main motions of Impα were described. Moreover, we determined the salt bridges, hydrogen bonds and hydrophobic interactions in the Impα-NplNLS interface. Our results show the bending and twisting motions participating in the recognition of nuclear proteins, allowing the accommodation and adjustment of a classical bipartite NLS sequence. The essential contacts for the nuclear import were also described and were mostly in agreement with previous studies, suggesting that the residues in the cNLS linker region establish important contacts with Impα adjusting the cNLS backbone. The MD simulations combined with NM analysis can be applied to the Impα-NLS system to help understand interactions between Impα and cNLSs and the analysis of non-classic NLSs.

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<![CDATA[Soaking suggests “alternative facts”: Only co-crystallization discloses major ligand-induced interface rearrangements of a homodimeric tRNA-binding protein indicating a novel mode-of-inhibition]]> https://www.researchpad.co/article/5989db52ab0ee8fa60bdc8f9

For the efficient pathogenesis of Shigella, the causative agent of bacillary dysentery, full functionality of tRNA-guanine transglycosylase (TGT) is mandatory. TGT performs post-transcriptional modifications of tRNAs in the anticodon loop taking impact on virulence development. This suggests TGT as a putative target for selective anti-shigellosis drug therapy. Since bacterial TGT is only functional as homodimer, its activity can be inhibited either by blocking its active site or by preventing dimerization. Recently, we discovered that in some crystal structures obtained by soaking the full conformational adaptation most likely induced in solution upon ligand binding is not displayed. Thus, soaked structures may be misleading and suggest irrelevant binding modes. Accordingly, we re-investigated these complexes by co-crystallization. The obtained structures revealed large conformational rearrangements not visible in the soaked complexes. They result from spatial perturbations in the ribose-34/phosphate-35 recognition pocket and, consequently, an extended loop-helix motif required to prevent access of water molecules into the dimer interface loses its geometric integrity. Thermodynamic profiles of ligand binding in solution indicate favorable entropic contributions to complex formation when large conformational adaptations in the dimer interface are involved. Native MS titration experiments reveal the extent to which the homodimer is destabilized in the presence of each inhibitor. Unexpectedly, one ligand causes a complete rearrangement of subunit packing within the homodimer, never observed in any other TGT crystal structure before. Likely, this novel twisted dimer is catalytically inactive and, therefore, suggests that stabilizing this non-productive subunit arrangement may be used as a further strategy for TGT inhibition.

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<![CDATA[Determining the role of missense mutations in the POU domain of HNF1A that reduce the DNA-binding affinity: A computational approach]]> https://www.researchpad.co/article/5989db53ab0ee8fa60bdccf0

Maturity-onset diabetes of the young type 3 (MODY3) is a non-ketotic form of diabetes associated with poor insulin secretion. Over the past years, several studies have reported the association of missense mutations in the Hepatocyte Nuclear Factor 1 Alpha (HNF1A) with MODY3. Missense mutations in the POU homeodomain (POUH) of HNF1A hinder binding to the DNA, thereby leading to a dysfunctional protein. Missense mutations of the HNF1A were retrieved from public databases and subjected to a three-step computational mutational analysis to identify the underlying mechanism. First, the pathogenicity and stability of the mutations were analyzed to determine whether they alter protein structure and function. Second, the sequence conservation and DNA-binding sites of the mutant positions were assessed; as HNF1A protein is a transcription factor. Finally, the biochemical properties of the biological system were validated using molecular dynamic simulations in Gromacs 4.6.3 package. Two arginine residues (131 and 203) in the HNF1A protein are highly conserved residues and contribute to the function of the protein. Furthermore, the R131W, R131Q, and R203C mutations were predicted to be highly deleterious by in silico tools and showed lower binding affinity with DNA when compared to the native protein using the molecular docking analysis. Triplicate runs of molecular dynamic (MD) simulations (50ns) revealed smaller changes in patterns of deviation, fluctuation, and compactness, in complexes containing the R131Q and R131W mutations, compared to complexes containing the R203C mutant complex. We observed reduction in the number of intermolecular hydrogen bonds, compactness, and electrostatic potential, as well as the loss of salt bridges, in the R203C mutant complex. Substitution of arginine with cysteine at position 203 decreases the affinity of the protein for DNA, thereby destabilizing the protein. Based on our current findings, the MD approach is an important tool for elucidating the impact and affinity of mutations in DNA-protein interactions and understanding their function.

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<![CDATA[Establishing knowledge on the sequence arrangement pattern of nucleated protein folding]]> https://www.researchpad.co/article/5989db50ab0ee8fa60bdbe4d

The heat-tolerance mechanisms of (hyper)thermophilic proteins provide a unique opportunity to investigate the unsolved protein folding problem. In an attempt to determine whether the interval between residues in sequence might play a role in determining thermostability, we constructed a sequence interval-dependent value function to calculate the residue pair frequency. Additionally, we identified a new sequence arrangement pattern, where like-charged residues tend to be adjacently assembled, while unlike-charged residues are distributed over longer intervals, using statistical analysis of a large sequence database. This finding indicated that increasing the intervals between unlike-charged residues can increase protein thermostability, with the arrangement patterns of these charged residues serving as thermodynamically favorable nucleation points for protein folding. Additionally, we identified that the residue pairs K-E, R-E, L-V and V-V involving long sequence intervals play important roles involving increased protein thermostability. This work demonstrated a novel approach for considering sequence intervals as keys to understanding protein folding. Our findings of novel relationships between residue arrangement and protein thermostability can be used in industry and academia to aid the design of thermostable proteins.

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