Proteins-Structure Function and Bioinformatics最新文献

SR/RS dipeptide repeats vary in both length and position, and are phosphorylated by SR protein kinases (SRPKs). PIM-1L, the long isoform of PIM-1 kinase, the splicing of which has been implicated in acute myeloid leukemia, contains a domain that consists largely of repeating SR/RS and SH/HS dipeptides (SR/SH-rich). In order to extend our knowledge on the specificity and cellular functions of SRPK1, here we investigate whether PIM-1L could act as substrate of SRPK1 by a combination of biochemical and computational approaches. Our biochemical data showed that the SR/SH-rich domain of PIM-1L was able to associate with SRPK1, yet it could not act as a substrate but, instead, inactivated the kinase. In line with our biochemical data, molecular modeling followed by a microsecond-scale all-atom molecular dynamics (MD) simulation suggests that the SR/SH-rich domain acts as a pseudo-docking peptide that binds to the same acidic docking-groove used in other SRPK1 interactions and induces inactive SRPK1 conformations. Comparative community network analysis of the MD trajectories, unraveled the dynamic architecture of apo SRPK1 and notable alterations of allosteric communications upon PIM-1L peptide binding. This analysis also allowed us to identify key SRPK1 residues, including unique ones, with a pivotal role in mediating allosteric signal propagation within the kinase core. Interestingly, most of the identified amino acids correspond to cancer-associated amino acid changes, validating our results. In total, this work provides insights not only on the details of SRPK1 inhibition by the PIM-1L SR/SH-domain, but also contributes to an in-depth understanding of SRPK1 regulation.

Alignments in bioinformatics refer to the arrangement of sequences to identify regions of similarity that can indicate functional, structural, or evolutionary relationships. They are crucial for bioinformaticians as they enable accurate predictions and analyses in various applications, including protein subcellular localization. The predictive model used in this article is based on a deep - convolutional architecture. We tested configurations of Deep N-to-1 convolutional neural networks of various depths and widths during experimentation for the evaluation of better-performing values across a diverse set of eight classes. For without alignment assessment, sequences are encoded using one-hot encoding, converting each character into a numerical representation, which is straightforward for non-numerical data and useful for machine learning models. For with alignments assessment, multiple sequence alignments (MSAs) are created using PSI-BLAST, capturing evolutionary information by calculating frequencies of residues and gaps. The average difference in peak performance between models with alignments and without alignments is approximately 15.82%. The average difference in the highest accuracy achieved with alignments compared with without alignments is approximately 15.16%. Thus, extensive experimentation indicates that higher alignment accuracy implies a more reliable model and improved prediction accuracy, which can be trusted to deliver consistent performance across different layers and classes of subcellular localization predictions. This research provides valuable insights into prediction accuracies with and without alignments, offering bioinformaticians an effective tool for better understanding while potentially reducing the need for extensive experimental validations. The source code and datasets are available at http://distilldeep.ucd.ie/SCL8/.

RfaH is a two-domain metamorphic protein involved in transcription regulation and translation initiation. To carry out its dual functions, RfaH relies on two coupled structural changes: Domain dissociation and fold switching. In the free state, the C-terminal domain (CTD) of RfaH adopts an all-α fold and is tightly associated with the N-terminal domain (NTD). Upon binding to RNA polymerase (RNAP), the domains dissociate and the CTD transforms into an all-β fold while the NTD remains largely, but not entirely, unchanged. We test the idea that a change in the conformation of an extended β-hairpin (β3-β4) located on the NTD, helps trigger domain dissociation. To this end, we use homology modeling to construct a structure, H₁, which is similar to free RfaH but with a remodeled β3-β4 hairpin. We then use an all-atom physics-based model enhanced with a dual basin structure-based potential to simulate domain separation driven by the thermal unfolding of the CTD with NTD in a fixed, folded conformation. We apply our model to both free RfaH and H₁. For H₁ we find, in line with our hypothesis, that the CTD exhibits lower stability and the domains dissociate at a lower temperature T, as compared to free RfaH. We do not, however, observe complete refolding to the all-β state in these simulations, suggesting that a change in β3-β4 orientation aids in, but is not sufficient for, domain dissociation. In addition, we study the reverse fold switch in which RfaH returns from a domain-open all-β state to its domain-closed all-α state. We observe a T-dependent transition rate; fold switching is slow at low T, where the CTD tends to be kinetically trapped in its all-β state, and at high-T, where the all-α state becomes unstable. Consequently, our simulations suggest an optimal T at which fold switching is most rapid. At this T, the stabilities of both folds are reduced. Overall, our study suggests that both inter-domain interactions and conformational changes within NTD may be important for the proper functioning of RfaH.

For a variety of applications, protein structures are clustered by sequence similarity, and sequence-redundant structures are disregarded. Sequence-similar chains are likely to have similar structures, but significant structural variation, as measured with RMSD, has been documented for sequence-similar chains and found usually to have a functional explanation. Moving two neighboring stretches of backbone through each other may change the chain topology and alter possible folding paths. The size of this motion is compatible to a variation in a flexible loop. We search and find domains with alternate chain topology in CATH4.2 sequence families relatively independent of sequence identity and of structural similarity as measured by RMSD. Structural, topological, and functional representative sets should therefore keep sequence-similar domains not just with structural variation but also with topological variation. We present BCAlign that finds Alignment and superposition of protein Backbone Curves by optimizing a user chosen convex combination of structural derivation and derivation between the structure-based sequence alignment and an input sequence alignment. Steric and topological obstructions from deforming a curve into an aligned curve are then found by a previously developed algorithm. For highly sequence-similar domains, sequence-based structural alignment better represents the chains motion and generally reveals larger structural and topological variation than structure-based does. Fold-switching protein pairs have been reported to be most frequent between X-ray and NMR structures and estimated to be underrepresented in the PDB as the alternate configuration is harder to resolve. Here we similarly find chain topology most frequently altered between X-ray and NMR structures.

The RAF1-RAP1A interaction activates the MAPK/ERK pathway which is very crucial in the carcinogenesis process. This protein complex influences tumor formation, proliferation, and metastasis. Understanding aberrant interactions driven by clinical mutations is vital for targeted therapies. Hence, the current study focuses on the screening of clinically reported substitutions in the RAF1 and RAP1A genes using predictive algorithms integrated with all-atoms simulation, essential dynamics, and binding free energy methods. Survival analysis results revealed a strong association between RAF1 and RAP1A expression levels and diminished survival rates in cancer patients across different cancer types. Integrated machine learning algorithms showed that among the 134 mutations reported for these 2 proteins, only 13 and 35 were classified as deleterious mutations in RAF1 and RAP1P, respectively. Moreover, one mutation in RAF1 reported elevated levels of binding between RAF1 and RAP1P while in RAP1A, 7 mutations were reported to increase the binding affinity. The high-binding mutations, P34Q and V60F, were subjected to protein-protein coupling which confirmed the increase in the binding affinity. Wild-type and mutant RAF1-RAP1P bound complexes were subjected to molecular simulation investigation, revealing enhanced structural stability, increased compactness, and stabilized residue fluctuations of the mutant systems in contrast to the wild-type. In addition, hydrogen bonding analysis revealed a variation in the binding paradigm which further underscores the impact of these substitutions on the coupling of RAF1 and RAP1A. Principal component analysis (PCA) and free energy landscape (FEL) evaluation further determined dynamical variations in the wild-type and mutant complexes. Finally, the Gibbs free energy for each complex was estimated and found to be -71.94 ± 0.38 kcal/mol for the wild-type, -95.57 ± 0.37 kcal/mol for the V60F, and -85.76 ± 0.72 kcal/mol for P34Q complex. These findings confirm the effect of these variants on increasing the binding affinity of RAF1 to RAP1P. These mutations can therefore be targeted for cancer therapy to modulate the activity of the MAPK/ERK signaling pathway.

Grp94 is the endoplasmic reticulum paralog of the hsp90 family of chaperones, which have been targeted for therapeutic intervention via their highly conserved ATP binding sites. The design of paralog-selective inhibitors relies on understanding the protein structural elements that drive higher affinity in selective inhibitors. Here, we determined the structures of Grp94 and Hsp90 in complex with the Grp94-selective inhibitor PU-H36, and of Grp94 with the non-selective inhibitor PU-H71. In Grp94, PU-H36 derives its higher affinity by utilizing Site 2, a Grp94-specific side pocket adjoining the ATP binding cavity, but in Hsp90 PU-H36 occupies Site 1, a side pocket that is accessible in all paralogs with which it makes lower affinity interactions. The structure of Grp94 in complex with PU-H71 shows only Site 1 binding. While changes in the conformation of helices 4 and 5 in the N-terminal domain occur when ligands bind to Site 1 of both Hsp90 and Grp94, large conformational shifts that also involve helix 1 are associated with the engagement of the Site 2 pocket in Grp94 only. Site 2 in Hsp90 is blocked and its helix 1 conformation is insensitive to ligand binding. To understand the role of helix 1 in ligand selectivity, we tested the binding of PU-H36 and other Grp94-selective ligands to chimeric Grp94/Hsp90 constructs. These studies show that helix 1 is the major determinant of selectivity for Site 2 targeted ligands and also influences the rate of ATPase activity in Hsp90 paralogs.

Biased agonism in G protein-coupled receptors is a phenomenon resulting in the selective activation of distinct intracellular signaling pathways by different agonists, which may exhibit bias toward either Gs, Gi, or arrestin-mediated pathways. This study investigates the structural basis of ligand-induced biased agonism within the context of the β₂-adrenergic receptor (β₂-AR). Atomistic molecular dynamics simulations were conducted for β₂-AR complexes with two stereoisomers of methoxynaphtyl fenoterol (MNFen), that is, compounds eliciting qualitatively different cellular responses. The simulations reveal distinct interaction patterns within the binding cavity, dependent on the stereoisomer. These changes propagate to the intracellular parts of the receptor, triggering various structural responses: the dynamic structure of the intracellular regions of the (R,R)-MNFen complex more closely resembles the "G_s-compatible" and "β-arrestin-compatible" conformation of β₂-AR, while both stereoisomers maintain structural responses equidistant from the inactive conformation. These findings are confirmed by independent coarse-grained simulations. In the context of deciphered molecular mechanisms, Trp313 plays a pivotal role, altering its orientation upon interactions with (R,R)-MNFen, along with the Lys305-Asp192 ionic bridge. This effect, accompanied by ligand interactions with residues on TM2, increases the strength of interactions within the extracellular region and the binding cavity, resulting in a slightly more open conformation and a minor (by ca. 0.2 nm) increase in the distance between the TM5-TM7, TM1-TM6, TM6-TM7, and TM1-TM5 pairs. On the other hand, an even slighter decrease in the distance between the TM1-TM4 and TM2-TM4 pairs is observed.

Protein cis-regulatory elements (CREs) are regions that modulate the activity of a protein through intramolecular interactions. Kinases, pivotal enzymes in numerous biological processes, often undergo regulatory control via inhibitory interactions in cis. This study delves into the mechanisms of cis regulation in kinases mediated by CREs, employing a combined structural and sequence analysis. To accomplish this, we curated an extensive dataset of kinases featuring annotated CREs, organized into homolog families through multiple sequence alignments. Key molecular attributes, including disorder and secondary structure content, active and ATP-binding sites, post-translational modifications, and disease-associated mutations, were systematically mapped onto all sequences. Additionally, we explored the potential for conformational changes between active and inactive states. Finally, we explored the presence of these kinases within membraneless organelles and elucidated their functional roles therein. CREs display a continuum of structures, ranging from short disordered stretches to fully folded domains. The adaptability demonstrated by CREs in achieving the common goal of kinase inhibition spans from direct autoinhibitory interaction with the active site within the kinase domain, to CREs binding to an alternative site, inducing allosteric regulation revealing distinct types of inhibitory mechanisms, which we exemplify by archetypical representative systems. While this study provides a systematic approach to comprehend kinase CREs, further experimental investigations are imperative to unravel the complexity within distinct kinase families. The insights gleaned from this research lay the foundation for future studies aiming to decipher the molecular basis of kinase dysregulation, and explore potential therapeutic interventions.

Porcine reproductive and respiratory syndrome (PRRS) is one of the most serious infectious immunosuppressive diseases in the world. The nonstructural protein Nsp4 can be used as an ideal target for anti-PRRSV replication inhibitors. However, little is known about potential inhibitors that target Nsp4 to affect PRRSV replication. The purpose of this study was to screen potential natural inhibitors that affect PRRSV replication by inhibiting Nsp4. Five compounds with strong binding affinity to Nsp4 were selected by structure-based molecular docking method. The complexes of naringin dihydrochalcone (NDC), agathisflavone (AGT), and amentoflavone (AMF) with Nsp4 were stable throughout the molecular dynamics simulation. According to MM/PBSA analysis, the free energies of binding of NDC, AGT, and AMF to Nsp4 were less than-30 Kcal/mol. In conclusion, these three compounds are worthy of further investigation as novel inhibitors of PRRSV. This study provides a theoretical basis for the development of anti-PRRSV natural drugs.

Microneme protein 2 (MIC2) and its associated protein M2AP are pivotal for the gliding motility and host cell invasion by Toxoplasma gondii. In our prior work, we showed that M2AP binds specifically to the sixth TSR domain of MIC2, with this interaction mediated dominantly by the hotspot residue H620 situated at the center of TSR6. To delve deeper into the functional significance of H620 and explore the dynamic behavior of Y602, we conducted molecular dynamic (MD) simulations of the Toxoplasma TSR6-M2AP complex, encompassing both wild-type and mutant forms. Our findings underscore the critical role of H620 within TSR6, particularly its hydrogen bond interaction with K72 of M2AP. The H620A mutation disrupts the nearby hydrophobic network while minimally affecting other hydrophilic interactions. Furthermore, our data reveal a highly conserved binding pose between M2AP and TSR6 across different species, consistent with previous trans-genera studies, thereby offering insights for future strategies in infection control development.