Proteins: Structure, Function, Bioinformatics
Brugia malayi is a parasitic nematode that causes lymphatic filariasis in humans. Here the solution structure of the forkhead DNA binding domain of Brugia malayi DAF-16a, a putative ortholog of Caenorhabditis elegans DAF-16, is reported. It is believed to be the first structure of a forkhead or winged helix domain from an invertebrate. C. elegans DAF-16 is involved in the insulin/IGF-I signaling pathway and helps control metabolism, longevity, and development. Conservation of sequence and structure with human FOXO proteins suggest that B. malayi DAF-16a is a member of the FOXO family of forkhead proteins. © Proteins 2014;. © 2014 Wiley Periodicals, Inc.
TGP, an extremely stable, non-aggregating fluorescent protein created by structure-guided surface engineering
In this paper we describe the engineering and X-ray crystal structure of Thermal Green Protein (TGP), an extremely stable, highly soluble, non-aggregating green fluorescent protein. TGP is a soluble variant of the fluorescent protein eCGP123, which despite being highly stable, has proven to be aggregation-prone. The X-ray crystal structure of eCGP123, also determined within the context of this paper, was used to carry out rational surface engineering to improve its solubility, leading to TGP. The approach involved simultaneously eliminating crystal lattice contacts while increasing the overall negative charge of the protein. Despite intentional disruption of lattice contacts and introduction of high entropy glutamate side chains, TGP crystallized readily in a number of different conditions and the X-ray crystal structure of TGP was determined to 1.9 Å resolution. The structural reasons for the enhanced stability of TGP and eCGP123 are discussed. We demonstrate the utility of using TGP as a fusion partner in various assays and significantly, in amyloid assays in which the standard fluorescent protein, EGFP, is undesirable because of aberrant oligomerization. © Proteins 2014;. © 2014 Wiley Periodicals, Inc.
Structure of human SNX10 reveals insights into its role in human autosomal recessive osteopetrosis
Sorting nexin 10 (SNX10), the unique member of the SNX family having vacuolation activity in cells, was shown to be involved in the development of autosomal recessive osteopetrosis (ARO) in recent genetic studies. However, the molecular mechanism of the disease-related mutations affecting the biological function of SNX10 is unclear. Here, we report the crystal structure of human SNX10 to 2.6Å resolution. The structure reveals that SNX10 contains the extended phox-homology domain we previously proposed. Our study provides the structural details of those disease-related mutations. Combined with the vacuolation study of those mutations, we found that Tyr32 and Arg51 are important for the protein stability and both play a critical role in vacuolation activity, while Arg16Leu may affect the function of SNX10 in osteoclast through protein–protein interactions. Proteins 2014. © 2014 Wiley Periodicals, Inc.
Polyanion binding accelerates the formation of stable and low-toxic aggregates of ALS-linked SOD1 mutant A4V
The toxic property thus far shared by both ALS-linked SOD1 variants and wild-type SOD1 is an increased propensity to aggregation. However, whether SOD1 oligomers or aggregates are toxic to cells remains to be well defined. Moreover, how the toxic SOD1 species are removed from intra- and extracellular environments also needs to be further explored. The DNA binding has been shown to be capable of accelerating the aggregatio\n of wild-type and oxidized SOD1 forms under acidic and neutral conditions. In this study, we explore the binding of DNA and heparin, two types of essential life polyanions, to A4V, an ALS-linked SOD1 mutant, under acidic conditions, and its consequences. The polyanion binding alters the A4V conformation, neutralizes its local positive charges, and increases its local concentrations along the polyanion chain, which are sufficient to lead to acceleration of the pH-dependent A4V aggregation. The accelerated aggregation, which is ascribed to the polyanion binding-mediated removal or shortening of the lag phase in aggregation, contributes to the formation of amorphous A4V nanoparticles. The prolonged incubation with polyanions not only results in the complete conversion of likely soluble toxic A4V oligomers into non- and low-toxic SDS-resistant aggregates, but also increases their stability. Although this is only an initial step toward reducing the toxicity of SOD1 mutants, the accelerating role of polyanions in protein aggregation might become one of the rapid pathways that remove toxic forms of SOD1 mutants from intra- and extracellular environments. Proteins 2014. © 2014 Wiley Periodicals, Inc.
Protein-spanning water networks and implications for prediction of protein–protein interactions mediated through hydrophobic effects
Hydrophobic effects, often conflated with hydrophobic forces, are implicated as major determinants in biological association and self-assembly processes. Protein–protein interactions involved in signaling pathways in living systems are a prime example where hydrophobic effects have profound implications. In the context of protein–protein interactions, a priori knowledge of relevant binding interfaces (i.e., clusters of residues involved directly with binding interactions) is difficult. In the case of hydrophobically mediated interactions, use of hydropathy-based methods relying on single residue hydrophobicity properties are routinely and widely used to predict propensities for such residues to be present in hydrophobic interfaces. However, recent studies suggest that consideration of hydrophobicity for single residues on a protein surface require accounting of the local environment dictated by neighboring residues and local water. In this study, we use a method derived from percolation theory to evaluate spanning water networks in the first hydration shells of a series of small proteins. We use residue-based water density and single-linkage clustering methods to predict hydrophobic regions of proteins; these regions are putatively involved in binding interactions. We find that this simple method is able to predict with sufficient accuracy and coverage the binding interface residues of a series of proteins. The approach is competitive with automated servers. The results of this study highlight the importance of accounting of local environment in determining the hydrophobic nature of individual residues on protein surfaces.Proteins 2014. © 2014 Wiley Periodicals, Inc.
Molecular dynamic studies on the impact of mutations on the structure, stability, and N-terminal orientation of annexin A1: Implications for membrane aggregation
Multiple MD simulations were performed for the full-length wild-type A1, the full length A1 mutations S27E and S27A, as well as the N-terminal peptide (AMVSEFLKQAWFIDNEEQEYIKTVKGS27KGGPGSAVSPYPTFN) of wild-type A1 and mutations S27E and S27A. The MD simulation trajectories of about 350 ns were generated and analyzed to examine the changes of core domain calcium binding affinity, core domain and N-terminal domain structures, and N-terminal domain orientation. Our results indicated that S27A and S27E mutations caused little changes on the calcium-binding affinity of the core domain of A1. However, the S27A mutation made the N-terminal domain of A1 less helical, and made the N-terminal domain migrate faster toward the core domain; these impacts on A1 are beneficial to the membrane aggregation process. On the contrary, the S27E mutation made the N-terminal domain of A1 more stable, and hindered the migration to the core domain; these changes on A1 are antagonistic for the membrane aggregation process. Our results using MD simulations provide an atomistic explanation for experimental observations that the S27E mutant showed a higher calcium concentration requirement and lower maximal extent of aggregation, while the wild-type and two mutants S27E and S27A required identical calcium concentrations for liposome binding. Proteins 2014. © 2014 Wiley Periodicals, Inc.
An Application of Information Theory to a Three-Body Coarse-Grained Representation of Proteins in the PDB: Insights into the Structural and Evolutionary Roles of Residues in Protein Structure
Knowledge-based methods for analyzing protein structures, such as statistical potentials, primarily consider the distances between pairs of bodies (atoms or groups of atoms). Considerations of several bodies simultaneously are generally used to characterize bonded structural elements or those in close contact with each other, but historically do not consider atoms that are not in direct contact with each other. In this report, we introduce an information-theoretic method for detecting and quantifying distance-dependent through-space multibody relationships between the sidechains of three residues. The technique introduced is capable of producing convergent and consistent results when applied to a sufficiently large database of randomly chosen, experimentally solved protein structures. The results of our study can be shown to reproduce established physico-chemical properties of residues as well as more recently discovered properties and interactions. These results offer insight into the numerous roles that residues play in protein structure, as well as relationships between residue function, protein structure, and evolution. The techniques and insights presented in this work should be useful in the future development of novel knowledge-based tools for the evaluation of protein structure. © Proteins 2014;. © 2014 Wiley Periodicals, Inc.
Terminal sialic acids on CD44 N-glycans can block hyaluronan binding by forming competing intramolecular contacts with arginine sidechains
Specific sugar residues and their linkages form the basis of molecular recognition for interactions of glycoproteins with other biomolecules. Seemingly small changes, like the addition of a single monosaccharide in the covalently attached glycan component of glycoproteins, can greatly affect these interactions. For instance, the sialic acid capping of glycans affects protein-ligand binding involved in cell–cell and cell–matrix interactions. CD44 is a single-pass transmembrane glycoprotein whose binding with its carbohydrate ligand hyaluronan (HA), an extracellular matrix component, mediates processes such as leukocyte homing, cell adhesion, and tumor metastasis. This binding is highly regulated by glycosylation of the N-terminal extracellular hyaluronan-binding domain (HABD); specifically, sialic acid capped N-glycans of HABD inhibit ligand binding. However, the molecular mechanism behind this sialic acid mediated regulation has remained unknown. Two of the five N-glycosyation sites of HABD have been previously identified as having the greatest inhibitory effect on HA binding, but only if the glycans contain terminal sialic acid residues. These two sites, Asn25 and Asn120, were chosen for in silico glycosylation in this study. Here, from extensive standard molecular dynamics simulations and biased simulations, we propose a molecular mechanism for this behavior based on spontaneously-formed charge-paired hydrogen bonding interactions between the negatively-charged sialic acid residues and positively-charged Arg sidechains known to be critically important for binding to HA, which itself is negatively charged. Such intramolecular hydrogen bonds would preclude associations critical to hyaluronan binding. This observation suggests how CD44 and related glycoprotein binding is regulated by sialylation as cellular environments fluctuate. Proteins 2014. © 2014 Wiley Periodicals, Inc.
Sequence and conformational preferences at termini of α-helices in membrane proteins: Role of the helix environment
α-helices are amongst the most common secondary structural elements seen in membrane proteins and are packed in the form of helix bundles. These α-helices encounter varying external environments (hydrophobic, hydrophilic) that may influence the sequence preferences at their N and C-termini. The role of the external environment in stabilization of the helix termini in membrane proteins is still unknown. Here we analyze α-helices in a high-resolution dataset of integral α-helical membrane proteins and establish that their sequence and conformational preferences differ from those in globular proteins. We specifically examine these preferences at the N and C-termini in helices initiating/terminating inside the membrane core as well as in linkers connecting these transmembrane helices. We find that the sequence preferences and structural motifs at capping (Ncap and Ccap) and near-helical (N’ and C’) positions are influenced by a combination of features including the membrane environment and the innate helix initiation and termination property of residues forming structural motifs. We also find that a large number of helix termini which do not form any particular capping motif are stabilized by formation of hydrogen bonds and hydrophobic interactions contributed from the neighboring helices in the membrane protein. We further validate the sequence preferences obtained from our analysis with data from an ultradeep sequencing study that identifies evolutionarily conserved amino acids in the rat neurotensin receptor. The results from our analysis provide insights for the secondary structure prediction, modeling and design of membrane proteins. © Proteins 2014;. © 2014 Wiley Periodicals, Inc.
The crystal structure of archaeal serine hydroxymethyltransferase reveals idiosyncratic features likely required to withstand high temperatures
Serine hydroxymethyltransferases (SHMTs) play an essential role in one-carbon unit metabolism and are employed in biomimetic reactions. We determined the crystal structure of free (apo) and PLP-bound (holo) SHMT from Methanocaldococcus jannaschii, the first from a hyperthermophile, from the archaea domain of life and that uses H4MPT as a cofactor, at 2.83 and 3.0 Å resolution, respectively. Idiosyncratic features were observed that are likely to contribute to structure stabilization. At the dimer interface, the C-terminal region folds in a unique fashion with respect to SHMTs from eubacteria and eukarya. At the active site, the conserved tyrosine does not make a cation-π interaction with an arginine like that observed in all other SHMT structures, but establishes an amide-aromatic interaction with Asn257, at a different sequence position. This asparagine residue is conserved and occurs almost exclusively in (hyper)thermophile SHMTs. This led us to formulate the hypothesis that removal of frustrated interactions (such as the Arg-Tyr cation-π interaction occurring in mesophile SHMTs) is an additional strategy of adaptation to high temperature. Both peculiar features may be tested by designing enzyme variants potentially endowed with improved stability for applications in biomimetic processes. © Proteins 2014;. © 2014 Wiley Periodicals, Inc.
Lipid-associated aggregate formation of superoxide dismutase-1 is initiated by membrane-targeting loops
Copper-Zinc superoxide dismutase 1 (SOD1) is a homodimeric enzyme that protects cells from oxidative damage. Hereditary and sporadic amyotrophic lateral sclerosis may be linked to SOD1 when the enzyme is destabilized through mutation or environmental stress. The cytotoxicity of demetallated or apo-SOD1 aggregates may be due to their ability to cause defects within cell membranes by co-aggregating with phospholipids. SOD1 monomers may associate with the inner cell membrane to receive copper ions from membrane-bound copper chaperones. But how apo-SOD1 interacts with lipids is unclear. We have used atomistic molecular dynamics simulations to reveal that flexible electrostatic and zinc-binding loops in apo-SOD1 dimers play a critical role in the binding of 1-octanol clusters and phospholipid bilayer, without any significant unfolding of the protein. The apo-SOD1 monomer also associates with phospholipid bilayer via its zinc-binding loop rather than its exposed hydrophobic dimerization interface. Our observed orientation of the monomer on the bilayer would facilitate its association with a membrane-bound copper chaperone. The orientation also suggests how membrane-bound monomers could act as seeds for membrane-associated SOD1 aggregation. Proteins 2014. © 2014 Wiley Periodicals, Inc.
Backbone dependency further improves side chain prediction efficiency in the Energy-based Conformer Library (bEBL)
Side chain optimization is an integral component of many protein modeling applications. In these applications, the conformational freedom of the side chains is often explored using libraries of discrete, frequently occurring conformations. Because side chain optimization can pose a computationally intensive combinatorial problem, the nature of these conformer libraries is important for ensuring efficiency and accuracy in side chain prediction. We have previously developed an innovative method to create a conformer library with enhanced performance. The Energy-based Library (EBL) was obtained by analyzing the energetic interactions between conformers and a large number of natural protein environments from crystal structures. This process guided the selection of conformers with the highest propensity to fit into spaces that should accommodate a side chain. Because the method requires a large crystallographic data-set, the EBL was created in a backbone-independent fashion. However, it is well established that side chain conformation is strongly dependent on the local backbone geometry, and that backbone-dependent libraries are more efficient in side chain optimization. Here we present the backbone-dependent EBL (bEBL), whose conformers are independently sorted for each populated region of Ramachandran space. The resulting library closely mirrors the local backbone-dependent distribution of side chain conformation. Compared to the EBL, we demonstrate that the bEBL uses fewer conformers to produce similar side chain prediction outcomes, thus further improving performance with respect to the already efficient backbone-independent version of the library. Proteins 2014. © 2014 Wiley Periodicals, Inc.
Accurate single-sequence prediction of solvent accessible surface area using local and global features
We present a new approach for predicting the Accessible Surface Area (ASA) using a General Neural Network (GENN). The novelty of the new approach lies in not using residue mutation profiles generated by multiple sequence alignments as descriptive inputs. Instead we use solely sequential window information and global features such as single-residue and two-residue compositions of the chain. The resulting predictor is both highly more efficient than sequence alignment-based predictors and of comparable accuracy to them. Introduction of the global inputs significantly helps achieve this comparable accuracy. The predictor, termed ASAquick, is tested on predicting the ASA of globular proteins and found to perform similarly well for so-called easy and hard cases indicating generalizability and possible usability for de-novo protein structure prediction. The source code and a Linux executables for GENN and ASAquick are available from Research and Information Systems at http://mamiris.com, from the SPARKS Lab at http://sparks-lab.org, and from the Battelle Center for Mathematical Medicine at http://mathmed.org. Proteins 2014. © 2014 Wiley Periodicals, Inc.
In the framework of the mean-field theory for the order parameter, which characterizes the degree of deviating the structure of a network-like macromolecule from its natural state under the action of the external force, its mechanical hysteresis is considered. Elastic hysterical properties of a macromolecule are studied as a function of the applied force, temperature and some other parameters controlling the viscous-elastic behavior of network-like macromolecules. Proteins 2014. © 2014 Wiley Periodicals, Inc.
Talin-driven inside-out activation mechanism of platelet αIIbβ3 integrin probed by multimicrosecond, all-atom molecular dynamics simulations
Platelet aggregation is the consequence of the binding of extracellular bivalent ligands such as fibrinogen and von Willebrand factor to the high affinity, active state of integrin αIIbβ3. This state is achieved through a so-called “inside-out” mechanism characterized by the membrane-assisted formation of a complex between the F2 and F3 subdomains of intracellular protein talin and the integrin β3 tail. Here, we present the results of multi-microsecond, all-atom molecular dynamics simulations carried on the complete transmembrane (TM) and C-terminal (CT) domains of αIIbβ3 integrin in an explicit lipid-water environment, and in the presence or absence of the talin-1 F2 and F3 subdomains. These large-scale simulations provide unprecedented molecular-level insights into the talin-driven inside-out activation of αIIbβ3 integrin. Specifically, they suggest a preferred conformation of the complete αIIbβ3 TM/CT domains in a lipid-water environment, and testable hypotheses of key intermolecular interactions between αIIbβ3 integrin and the F2/F3 domains of talin-1. Notably, not only do these simulations give support to a stable left-handed reverse turn conformation of the αIIb juxtamembrane motif rather than a helical turn, but they raise the question as to whether TM helix separation is required for talin-driven integrin activation. Proteins 2014. © 2014 Wiley Periodicals, Inc.
Many mutations in the N-terminal arm of AraC result in constitutive behavior in which transcription of the araBAD genes occurs even in the absence of arabinose. To begin to understand the mechanism underlying this class of mutations, we used molecular dynamics with self-guided Langevin dynamics to simulate (1) wild type AraC, (2) known constitutive mutants resulting from alterations in the regulatory arm, particularly alanine and glycine substitutions at residue eight because P8G is constitutive whereas P8A behaves like wild type, and (3) selected variant AraC proteins containing alterations in the dimerization core. In all of the constitutive arm mutants, but not the wild type protein, residues 37-42, which are located in the core of the dimerization domain, became restructured. This raised the question of whether or not these structural changes are an obligatory component of constitutivity. Using molecular dynamics, we identified alterations in the core that produced a similar restructuring. The corresponding mutants were constructed and their ara constitutivity status was determined experimentally. Because the core mutants were not found to be constitutive, we conclude that restructuring of core residues 37-42 does not, itself, lead to constitutivity of AraC. The available data lead to the hypothesis that the interaction of the N-terminal arm with something other than the front lip is the primary determinant of the inducing versus repressing state of AraC. © Proteins 2014;. © 2014 Wiley Periodicals, Inc.
Alanine and proline content modulate global sensitivity to discrete perturbations in disordered proteins
Molecular transduction of biological signals is understood primarily in terms of the cooperative structural transitions of protein macromolecules, providing a mechanism through which discrete local structure perturbations affect global macromolecular properties. The recognition that proteins lacking tertiary stability, commonly referred to as intrinsically disordered proteins, mediate key signaling pathways suggests that protein structures without cooperative intramolecular interactions may also have the ability to couple local and global structure changes. Presented here are results from experiments that measured and tested the ability of disordered proteins to couple local changes in structure to global changes in structure. Using the intrinsically disordered N-terminal region of the p53 protein as an experimental model, a set of proline and alanine to glycine substitution variants were designed to modulate backbone conformational propensities without introducing non-native intramolecular interactions. The hydrodynamic radius (Rh) was used to monitor changes in global structure. Circular dichroism spectroscopy showed that the glycine substitutions decreased polyproline II (PPII) propensities relative to the wild type, as expected, and fluorescence methods indicated that substitution-induced changes in Rh were not associated with folding. The experiments showed that changes in local PPII structure cause changes in Rh that are variable and that depend on the intrinsic chain propensities of proline and alanine residues, demonstrating a mechanism for coupling local and global structure changes. Molecular simulations that model our results were used to extend the analysis to other proteins and illustrate the generality of the observed proline and alanine effects on the structures of intrinsically disordered proteins. © Proteins 2014;. © 2014 Wiley Periodicals, Inc.
Hamiltonian replica exchange combined with elastic network analysis to enhance global domain motions in atomistic molecular dynamics simulations
Coarse-grained elastic network models (ENM) of proteins offer a low resolution representation of protein dynamics and directions of global mobility. A Hamiltonian-replica exchange molecular dynamics (H-REMD) approach has been developed that combines information extracted from an ENM analysis with atomistic explicit solvent MD simulations. Based on a set of centers representing rigid segments (centroids) of a protein a distance-dependent biasing potential is constructed by means of an ENM analysis to promote and guide centroid/domain rearrangements. The biasing potentials are added with different magnitude to the force field description of the MD simulation along the replicas with one reference replica under the control of the original force field. The magnitude and the form of the biasing potentials are adapted during the simulation based on the average sampled conformation to reach a near constant biasing in each replica after equilibration. This allows for canonical sampling of conformational states in each replica. The application of the methodology to a 2-domain segment of the glycoprotein 130 and to the protein cyanovirin-N indicates significantly enhanced global domain motions and improved conformational sampling compared to conventional MD simulations. © Proteins 2014;. © 2014 Wiley Periodicals, Inc.
Computational enzyme design is an emerging field that has yielded promising success stories, but where numerous challenges remain. Accurate methods to rapidly evaluate possible enzyme design variants could provide significant value when combined with experimental efforts by reducing the number of variants needed to be synthesized and speeding the time to reach the desired endpoint of the design. To that end, extending our computational methods to model the fundamental physical-chemical principles that regulate activity in a protocol that is automated and accessible to a broad population of enzyme design researchers is essential. Here, we apply a physics-based implicit solvent MM-GBSA scoring approach to enzyme design and benchmark the computational predictions against experimentally determined activities. Specifically, we evaluate the ability of MM-GBSA to predict changes in affinity for a steroid binder protein, catalytic turnover for a Kemp eliminase, and catalytic activity for α-Gliadin peptidase variants. Using the enzyme design framework developed here, we accurately rank the most experimentally active enzyme variants, suggesting that this approach could provide enrichment of active variants in real-world enzyme design applications. © Proteins 2014;. © 2014 Wiley Periodicals, Inc.