Cationic helical peptides play a crucial role in applications such as anti-microbial and anti-cancer activity. The activity of these peptides directly correlates with their helicity. In this study, we have performed extensive all-atom molecular dynamics simulations of 25 Lysine-Leucine co-polypeptide sequences of varying charge density ( λ ) and patterns. Our findings showed that an increase in the charge density on the peptide leads to a gradual decrease in the helicity up to a critical charge density λ c . Beyond, λ c a complete helix to coil transition was observed. The decrease in the helicity correlated with the increased number of water molecules in first solvation shell, solvent-exposed surface area, and a higher value of the radius of gyration of the peptide.
Various post translational modifications like hyper phosphorylation, O-GlycNAcylation, and acetylation have been attributed to induce the abnormal folding in tau protein. Recent in vitro studies revealed the possible involvement of N–glycosylation of tau protein in the abnormal folding and tau aggregation. Hence in this study, we performed microsecond long all atom molecular dynamics simulation to gain insights into the effects of N-glycosylation on Asn-359residue which forms part of the microtubule binding region. Trajectory analysis of the stimulations coupled with essential dynamics and free energy landscape analysis suggested that tau, in its N-glycosylated form tend to exist in a largely folded conformation having high beta sheet propensity as compared to unmodified tau which exists in a large extended form with very less beta sheet propensity. Residue interaction network analysis of the lowest energy conformations further revealed that Phe378 and Lys353 are the functionally important residues in the peptide which helped in initiating the folding process and Phe378, Lys347&Lys370 helped maintaining the stability of the protein in the folded state.
Arrestins are important scaffolding proteins that are expressed in all vertebrate animals. They regulate cell signaling events upon binding to active G-protein coupled receptors ( GPCR) and trigger endocytosis of active GPCRs. While many of the functional sites on arrestins have been characterized, the question of how these sites interact is unanswered. We used anisotropic network modelling ( ANM) together with our covariance compliment techniques to survey all of the available structures of the non-visual arrestins to map how structural changes and protein-binding affect their structural dynamics. We found that activation and clathrin binding have a marked effect on arrestin dynamics, and that these dynamics changes are localized to a small number of distant functional sites. These sites include α-helix 1, the lariat loop, nuclear localization domain, and the C-domain β-sheets on the C-loop side. Our techniques suggest that clathrin binding and/or GPCR activation of arrestin perturb the dynamics of these sites independent of structural changes.
The inversion from L- to D-stereochemistry endows peptides improved bioactivity and enhanced resistance to many proteases and peptidases. To strengthen the biostability and bioavailability of peptide drugs, enzymatic epimerization becomes an important way to incorporate D-amino acid into peptide backbones. Recently, a bifunctional thioesterase NocTE, which is responsible for the epimerization and hydrolysis of the C-terminal (p-hydroxyphenyl)glycine residue of β-lactam antibiotic nocardicin A, exclusively directs to the generation of D-diastereomers. Different from other epimerases, NocTE exhibits unique stereochemical selectivity. Herein, we investigated the catalytic mechanism of NocTE via molecular dynamic (MD) simulations and quantum mechanical/molecular mechanics (QM/MM) calculations. Through structural analyses, two key water molecules around the reaction site were found to serve as proton mediators in epimerization. The structural characteristics inspired us to propose a substrate-assisted mechanism for the epimerization, where multi-step proton transfers were mediated by water molecules and β-lactam ring, and the free energy barrier was calculated to be 20.3 kcal/mol. After that, the hydrolysis of D-configured substrate was energetically feasible with the energy barrier of 14.3 kcal/mol. As a comparison, the energy barrier for the direct hydrolysis of L-configured substrate was obtained to be 24.0 kcal/mol. Our study provides mechanistic insights into catalytic activities of bifunctional thioesterase NocTE, uncovers more clues to the molecular basis for stereochemical selectivity and paves the way for the directed biosynthesis of novel peptide drugs with various stereostructural characteristics by enzyme rational design.
Mutations are the cause of several diseases as well as the underlying force of evolution. A thorough understanding of its biophysical consequences is essential. We present a computational framework for evaluating different levels of mutual information (MI) and its dependence on mutation. We used molecular dynamics trajectories of the third PDZ domain and its different mutations. MI calculated from these trajectories shows that: (i) the multivariate Gaussian distribution of joint probabilities characterizes the MI between residue pairs with sufficient accuracy. Nonlinearities in joint probabilities calculated by tensor Hermite polynomials up to the fifth order contribute insignificantly. (ii) Changes in MI between residue pairs show the characteristic patterns resulting from specific mutations. (iii) Triple correlations are characterized by evaluating MI between triplets of residues, certain triplets are strongly affected by mutation. (iv) Susceptibility of residues to perturbation are obtained by MI and discussed in terms of linear response theory.
The revelation of protein folding is a challenging subject in both discovery and description. Except acquirement of accurate 3D structure for protein stable state, another big hurdle is how to discover structural flexibility for protein innate character. Even if a huge number of flexible conformations are known, difficulty is how to describe these conformations. A novel approach, protein structure fingerprint, has been developed to expose the comprehensive local folding variations, and then construct folding conformations for entire protein. The backbone of 5 amino acid residues was identified as a universal folden, and then a set of Protein Folding Shape Code (PFSC) was derived for completely covering folding space in alphabetic description. Sequentially, a database was created to collect all possible folding shapes of local folding variations for all permutation of 5 amino acids. Successively, Protein Folding Variation Matrix (PFVM) assembled all possible local folding variations along sequence for a protein, which possesses several prominent features. First, it showed the fluctuation with certain folding patterns along sequence which revealed how the protein folding was related the order of amino acids in sequence. Second, all folding variations for an entire protein can be simultaneously apprehended at a glance within PFVM. Third, all conformations can be determined by local folding variations from PFVM, so total number of conformations is no longer ambiguous for any protein. Finally, the most possible folding conformation and its 3D structure can be acquired according PFVM for protein structure prediction. Therefore, the protein structure fingerprint approach provides a significant means for investigation of protein folding problem.
Myeloid cell leukemia-1 (MCL1), an anti-apoptotic BCL-2 family protein plays a major role in the control of apoptosis as the regulator of mitochondrial permeability which is deregulated in various solid and hematological malignancies. Interaction of the executioner proteins Bak/Bax with anti-apoptotic MCL1 and its cellular composition determines the apoptotic or survival pathway. This study highlighted the deleterious MCL1-Bax stabilizing effect of the mutation V220F on MCL1 structure through computational protein-protein interaction predictions and molecular dynamics simulations. The single point mutation at V220F was selected as it is residing at the hydrophobic core region of BH3 conserved domain, the site of Bax binding. The molecular dynamics simulation studies showed increase in stability of the mutated MCL1 before and after Bax binding comparable with the native MCL1. The clusters from free energy landscape found out structural variation in folding pattern with additional helix near the BH3 domain in the mutated structure. This loop to helix structural change in the mutated complex favored stable interaction of the complex and also induced Bax conformational change. Moreover, molecular mechanics based binding free energy calculations confirmed increased affinity of Bax towards mutated MCL1. Residue-wise interaction network analysis showed the individual residues in Bax binding responsible for the change in stability and interaction due to the protein mutation. In conclusion, the overall findings from the study reveal that the presence of V220F mutation on MCL1 is responsible for the structural confirmational change leading to disruption of its biological functions which might be responsible for tumorigenesis. The mutation could possibly be used as future diagnostic markers in treating cancers.
Dissimilatory sulfite reductase is an ancient enzyme that has linked the global sulfur and carbon biogeochemical cycles since at least 3.47 Gya. While much has been learned about the phylogenetic distribution and diversity of DsrAB across environmental gradients, far less is known about the structural changes that occurred to maintain DsrAB function as the enzyme accompanied diversification of sulfate/sulfite reducing organisms (SRO) into new environments. Analyses of available crystal structures of DsrAB from Archaeoglobus fulgidus and Desulfovibrio vulgaris, representing early and late evolving lineages, respectively, show that certain features of DsrAB are structurally conserved, including active siro-heme binding motifs. Whether such structural features are conserved among DsrAB recovered from varied environments, including hot spring environments that host representatives of the earliest evolving SRO lineage (e.g., MV2-Eury), is not known. To begin to overcome these gaps in our understanding of the evolution of DsrAB, structural models from MV2.Eury were generated and evolutionary sequence co-variance analyses were conducted on a curated DsrAB database. Phylogenetically diverse DsrAB harbor many conserved functional residues including those that ligate active siro-heme(s). However, evolutionary co-variance analysis of monomeric DsrAB subunits revealed several False Positive Evolutionary Couplings (FPEC) that correspond to residues that have co-evolved despite being too spatially distant in the monomeric structure to allow for direct contact. One set of FPECs corresponds to residues that form a structural path between the two active siro-heme moieties across the interface between heterodimers, suggesting the potential for allostery or electron transfer within the enzyme complex. Other FPECs correspond to structural loops and gaps that may have been selected to stabilize enzyme function in different environments. These structural bioinformatics results suggest that DsrAB has maintained allosteric communication pathways between subunits as SRO diversified into new environments. The observations outlined here provide a framework for future biochemical and structural analyses of DsrAB to examine potential allosteric control of this enzyme.
The c subunits, which constitutes the c-ring apparatus of the F 1F O-ATPase, could be the main components of the mitochondrial permeability transition pore (mPTP). The well-known modulator of the mPTP formation and opening is the cyclophilin D (CyPD), a peptidyl-prolyl cis- trans isomerase. On the loop, which connects the two hairpin α-helix of c subunit, is present the unique proline residue (Pro 40) that could be a biological target of CyPD. Indeed, the proline cis- trans isomerization might provide the switch that interconverts the open/closed states of the pore by pulling out the c-ring lipid plug.
The heterologous overexpression states of prion proteins play a critical role in understanding the mechanisms of prion-related diseases. We report herein the identification of soluble monomer and complex states for a bakers’ yeast prion, Sup35, when expressed in E. coli. Two peaks are apparent with the elution of His-tagged Sup35 by imidazole from a Ni 2+ affinity column. Peak I contains Sup35 in both monomer and aggregated states. Sup35 aggregate is abbreviated as C-aggregate and includes a non-fibril complex comprising Sup35 aggregate-HSP90-Dna K, ATP synthase β unit (chain D), 30S ribosome subunit, and Omp F. The purified monomer and C-aggregate can remain stable for an extended period of time. Peak II contains Sup35 also in both monomer and aggregated (abbreviated as S-aggregate) states, but the aggregated states are caused by the formation of inter-Sup35 disulfide bonds. This study demonstrates that further assembly of Sup35 non-fibril C-aggregate can be interrupted by the chaperone repertoire system in E. coli.
Petroleum based plastics are durable and accumulate in all ecological niches. Knowledge on enzymatic degradation is sparse. Today, less than 50 verified plastics-active enzymes are known. First examples of enzymes acting on the polymers polyethylene terephthalate (PET) and polyurethane (PUR) have been reported together with a detailed biochemical and structural description. Further, very few polyamide (PA) oligomer active enzymes are known. In this paper, the current known enzymes acting on the synthetic polymers PET and PUR are briefly summarized, their published activity data were collected and integrated into a comprehensive open access database. The Plastics-Active Enzymes Database (PAZy) represents an inventory of known and experimentally verified plastics-active enzymes. Almost 3000 homologues of PET-active enzymes were identified by profile hidden Markov models. Over 2000 homologues of PUR-active enzymes were identified by BLAST. Based on multiple sequence alignments, conservation analysis identified the most conserved amino acids, and sequence motifs for PET- and PUR-active enzymes were derived.
Functional regulation via conformational dynamics is well known in structured proteins, but less well characterized in intrinsically disordered proteins and their complexes. Using NMR spectroscopy we have identified a dynamic regulatory mechanism in the human insulin-like growth factor (IGF) system involving the central, intrinsically disordered linker domain of human IGF-binding protein-2 ( hIGFBP2). The bioavailability of IGFs is regulated by the proteolysis of IGF-binding proteins. In the case of hIGFBP2, the linker domain (L- hIGFBP2) retains its intrinsic disorder upon binding IGF-1 but its dynamics are significantly altered, both in the IGF binding region and distantly located protease cleavage sites. The increase in flexibility of the linker domain upon IGF-1 binding may explain the IGF-dependent modulation of proteolysis of IGFBP2 in this domain. As IGF homeostasis is important for cell growth and function, and its dysregulation is a key contributor to several cancers, our findings open up new avenues for the design of IGFBP analogs inhibiting IGF-dependent tumors.
Nucleotides metabolism is a fundamental process in all organisms. Two families of nucleoside phosphorylases (NP) that catalyze the phosphorolytic cleavage of the glycosidic bond in nucleosides have been found, including the trimeric or hexameric NP-I and dimeric NP-II family enzymes. Recently studies revealed another class of NP protein in E. coli named Pyrimidine/purine nucleoside phosphorylase (ppnP), which can catalyze the phosphorolysis of diverse nucleosides and yield D-ribose 1-phosphate and the respective free bases. Here, we solve the crystal structures of ppnP from E. coli and the other three species. Our studies revealed that the structure of ppnP belongs to the Rlmc-like cupin fold and showed as a rigid dimeric conformation. Detail analysis revealed a potential nucleoside binding pocket full of hydrophobic residues. And the residues involved in the dimer and pocket formation are all well conserved in bacteria. Since the cupin fold is a large superfamily in the biosynthesis of natural products, our studies provide the structural basis for understanding and the directed evolution of NP proteins.
The structure of a protein plays a pivotal role in determining its function. Often, the protein surface’s shape and curvature dictate its nature of interaction with other proteins and biomolecules. However, marked by corrugations and roughness, a protein’s surface representation poses significant challenges for its curvature-based characterization. In the present study, we employ unsupervised machine learning to segment the protein surface into patches. To measure the surface curvature of a patch, we present an algebraic sphere fitting method that is fast, accurate, and robust. Moreover, we use local curvatures to show the existence of “shape complementarity” in protein-protein, antigen-antibody, and protein-ligand interfaces. We believe that the current approach could help understand the relationship between protein structure and its biological function and can be used to find binding partners of a given protein.
Multimeric protein complexes are molecular apparatuses to regulate biological systems and often determine their fate. Among proteins forming such molecular assemblies, amyloid proteins have drawn attention over a half-century since amyloid fibril formation of these proteins is supposed to be a common pathogenic cause for neurodegenerative diseases. This process is triggered by the accumulation of fibril-like aggregates, while the microscopic mechanisms are mostly elusive due to technical limitation of experimental methodologies in individually observing each of diverse aggregate species in the aqueous solution. We then addressed this problem by employing atomistic molecular dynamics simulations for the paradigmatic amyloid protein, amyloid-β (1-42) (Aβ 42). Seven different dimeric forms of oligomeric Aβ 42 fibril-like aggregate in aqueous solution, ranging from tetramer to decamer, were considered. We found additive effects of the size of these fibril-like aggregates on their thermodynamic stability and have clarified kinetic suppression of protomer-protomer dissociation reactions at and beyond the point of pentamer dimer formation. This observation was obtained from the specific combination of the Aβ 42 protomer structure and the physicochemical condition that we here examined, while it is worthwhile to recall that several amyloid fibrils take dimeric forms of their protomers. We could thus conclude that the stable formation of fibril-like protomer dimer should be involved in a turning point where rapid growth of amyloid fibrils is triggered.
RNA binding proteins (RBPs) regulate many important cellular processes through their interactions with RNA molecules. RBPs are critical for post-transcriptional mechanisms keeping gene regulation in a fine equilibrium. Conversely, dysregulation of RBPs and RNA metabolism pathways is an established hallmark of tumorigenesis. Human nucleolin (NCL) is a multifunctional RBP that interacts with different types of RNA molecules, in part through its four RNA binding domains (RBDs). Particularly, NCL interacts directly with microRNAs (miRNAs) and is involved in their aberrant processing linked with many cancers, including breast cancer. Nonetheless, molecular details of the NCL-miRNA interaction remain obscure. In this study, we used an in silico approach to characterize how NCL targets miRNAs and whether this specificity is imposed by a definite RBD-interface. Here, we present structural models of NCL-RBDs and miRNAs, as well as predict scenarios of NCL- miRNA interactions generated using docking algorithms. Our study suggests a predominant role of NCL RBDs 3 and 4 (RBD3-4) in miRNA binding. We provide detailed analyses of specific motifs/residues at the NCL-substrate interface in both these RBDs and miRNAs. Finally, we propose that the evolutionary emergence of more than two RBDs in NCL in higher organisms coincides with its additional role/s in miRNA processing. Our study shows that RBD3-4 display sequence/structural determinants to specifically recognize miRNA precursor molecules. Moreover, the insights from this study can ultimately support the design of novel antineoplastic drugs aimed at regulating NCL-dependent biological pathways with a causal role in tumorigenesis.
Lipid transporters play an important role in most if not all organisms, ranging from bacteria to humans. For example, in Mycobacterium tuberculosis, the trehalose monomycolate transporter MmpL3 is involved in cell wall biosynthesis, while in humans, cholesterol transporters are involved in normal cell function as well as in disease. Here, using structural and bioinformatics information, we propose that there are proteins that also contain “MmpL3-like” (MMPL) transmembrane (TM) domains in many protozoa, including Trypanosoma cruzi, as well as in the bacterium Staphylococcus aureus, where the fatty acid transporter FarE has the same set of “active-site” residues as those found in the mycobacterial MmpL3s, and in T. cruzi. We also show that there are strong sequence and predicted structural similarities between the TM proton-translocation domain seen in the X-ray structures of mycobacterial MmpL3s and several human as well as fungal lipid transporters, leading to the proposal that there are similar proteins in apicomplexan parasites, and in plants. The animal, fungal, apicomplexan and plant proteins have larger extra-membrane domains than are found in the bacterial MmpL3, but they have a similar TM domain architecture, with the introduction of a (catalytically essential) Phe>His residue change, and a Ser/Thr H-bond network, involved in H +-transport. Overall, the results are of interest since they show that MMPL-family proteins are present in essentially all life-forms: archaea, bacteria, protozoa, fungi, plants and animals and, where known, they are involved in “lipid” (glycolipid, phospholipid, sphingolipid, fatty acid, cholesterol, ergosterol) transport, powered by transmembrane molecular pumps having similar structures.
In Azospirillum brasilense, an extra-cytoplasmic function sigma factor (RpoE10) shows the characteristic 119 amino acid long C-terminal extension found in ECF41-type sigma factors, which possesses three conserved motifs (WLPEP, DGGGR, and NPDKV), one in the linker region between the sigma 2 and sigma 4, and the other two in the SnoaL_2 domain of the C-terminal extension. Here, we have described the role of the two conserved motifs in the SnoaL_2 domain of RpoE10 in the inhibition and activation of its activity, respectively. Truncation of the distal part of the C-terminal sequence of the RpoE10 (including NPDKV but excluding the DGGGR motif) results in its promoter’s activation suggesting autoregulation. Further truncation of the C-terminal sequence up to its proximal part, including NPDKV and DGGGR motif, abolished promoter activation. Replacement of NPDKV motif with NAAAV in RpoE10 increased its ability to activate its promoter, whereas replacement of DGGGR motif led to reduced promoter activation. We have explored the dynamic modulation of sigma2 – sigma4 domains and the relevant molecular interactions mediated by the two conserved motifs of the SnoaL2 domain using molecular dynamics simulation. The analysis enabled us to explain that the NPDKV motif located distally in the C-terminus negatively impacts transcriptional activation. In contrast, the DGGGR motif found proximally of the C-terminal extension is required to activate RpoE1