Coiled‐coil interactions are required for post‐Golgi R‐SNARE trafficking

The sorting of post‐Golgi R‐SNAREs (vesicle‐associated membrane protein (VAMP)1, 2, 3, 4, 7 and 8) is still poorly understood. To address this, we developed a system to investigate their localization, trafficking and cell‐surface levels. Here, we show that the distribution and internalization of VAMPs 3 and 8 are determined solely through a new conserved mechanism that uses coiled‐coil interactions, and that VAMP4 does not require these interactions for its trafficking. We propose that VAMPs 3 and 8 are trafficked while in a complex with Q‐SNAREs. We also show that the dileucine motif of VAMP4 is required for both its internalization and retrieval to the trans‐Golgi network. However, when the dileucine motif is mutated, the construct can still be internalized potentially through coiled‐coil interactions with Q‐SNAREs.     

Two‐domain analysis of JmjN‐JmjC and PHD‐JmjC lysine demethylases: Detecting an inter‐domain evolutionary stress

Residues at different positions of a multiple sequence alignment sometimes evolve together, due to a correlated structural or functional stress at these positions. Co‐evolution has thus been evidenced computationally in multiple proteins or protein domains. Here, we wish to study whether an evolutionary stress is exerted on a sequence alignment across protein domains, i.e., on longer sequence separations than within a single protein domain. JmjC‐containing lysine demethylases were chosen for analysis, as a follow‐up to previous studies; these proteins are important multidomain epigenetic regulators. In these proteins, the JmjC domain is responsible for the demethylase activity, and surrounding domains interact with histones, DNA or partner proteins. This family of enzymes was analyzed at the sequence level, in order to determine whether the sequence of JmjC‐domains was affected by the presence of a neighboring JmjN domain or PHD finger in the protein. Multiple positions within JmjC sequences were shown to have their residue distributions significantly altered by the presence of the second domain. Structural considerations confirmed the relevance of the analysis for JmjN‐JmjC proteins, while among PHD‐JmjC proteins, the length of the linker region could be correlated to the residues observed at the most affected positions. The correlation of domain architecture with residue types at certain positions, as well as that of overall architecture with protein function, is discussed. The present results thus evidence the existence of an across‐domain evolutionary stress in JmjC‐containing demethylases, and provide further insights into the overall domain architecture of JmjC domain‐containing proteins.     

Ab initio folding of a trefoil‐fold motif reveals structural similarity with a β‐propeller blade motif

Many protein architectures exhibit evidence of internal rotational symmetry postulated to be the result of gene duplication/fusion events involving a primordial polypeptide motif. A common feature of such structures is a domain‐swapped arrangement at the interface of the N‐ and C‐termini motifs and postulated to provide cooperative interactions that promote folding and stability. De novo designed symmetric protein architectures have demonstrated an ability to accommodate circular permutation of the N‐ and C‐termini in the overall architecture; however, the folding requirement of the primordial motif is poorly understood, and tolerance to circular permutation is essentially unknown. The β‐trefoil protein fold is a threefold‐symmetric architecture where the repeating ~42‐mer “trefoil‐fold” motif assembles via a domain‐swapped arrangement. The trefoil‐fold structure in isolation exposes considerable hydrophobic area that is otherwise buried in the intact β‐trefoil trimeric assembly. The trefoil‐fold sequence is not predicted to adopt the trefoil‐fold architecture in ab initio folding studies; rather, the predicted fold is closely related to a compact “blade” motif from the β‐propeller architecture. Expression of a trefoil‐fold sequence and circular permutants shows that only the wild‐type N‐terminal motif definition yields an intact β‐trefoil trimeric assembly, while permutants yield monomers. The results elucidate the folding requirements of the primordial trefoil‐fold motif, and also suggest that this motif may sample a compact conformation that limits hydrophobic residue exposure, contains key trefoil‐fold structural features, but is more structurally homologous to a β‐propeller blade motif.     

Assessing the genetic diversity of Panicum coloratum var. makarikariense using agro‐morphological traits and microsatellite‐based markers

Panicum coloratum var. makarikariense is a perennial C₄ grass native to South Africa with relatively good forage production under limited‐resource conditions. Genetic characterisation and breeding efforts have been scant, thus limiting its use in cattle raising systems. The goal of the present study was to assess the genetic diversity of a collection of P. coloratum var. makarikariense using agro‐morphological traits and molecular markers, in comparison with one accession of var. coloratum and one population of Panicum bergii. Agro‐morphological variability between and within accessions of var. makarikariense in a common garden setting was observed, showing that there is still opportunity for selection. Some accessions performed better than the commercialised material in relation to potential forage production. A total of 117 ISSR bands and 48 SSR alleles allowed the detection of genetic variability between and within accessions. The presence of accession‐specific bands suggested distinctness and limited gene flow. The genetic variability encountered in the commercialised material suggested that it is a stabilised population which has not undergone a strong selection process. Low correlation between agro‐morphologic and molecular variability was observed indicating that both approaches provide complementary information. Both morphological and molecular markers reveal genetic differentiation between varieties and species. This study provides a set of new SSR markers available for diversity assessment and valuable information that can be applied directly in collection management for breeding and conservation programmes.     

Clustering of multi‐domain protein sequences

The overall function of a multi‐domain protein is determined by the functional and structural interplay of its constituent domains. Traditional sequence alignment‐based methods commonly utilize domain‐level information and provide classification only at the level of domains. Such methods are not capable of taking into account the contributions of other domains in the proteins, and domain‐linker regions and classify multi‐domain proteins. An alignment‐free protein sequence comparison tool, CLAP (CLAssification of Proteins) was previously developed in our laboratory to especially handle multi‐domain protein sequences without a requirement of defining domain boundaries and sequential order of domains. Through this method we aim to achieve a biologically meaningful classification scheme for multi‐domain protein sequences. In this article, CLAP‐based classification has been explored on 5 datasets of multi‐domain proteins and we present detailed analysis for proteins containing (1) Tyrosine phosphatase and (2) SH3 domain. At the domain‐level CLAP‐based classification scheme resulted in a clustering similar to that obtained from an alignment‐based method. CLAP‐based clusters obtained for full‐length datasets were shown to comprise of proteins with similar functions and domain architectures. Our study demonstrates that multi‐domain proteins could be classified effectively by considering full‐length sequences without a requirement of identification of domains in the sequence.     

Oligomerization of a symmetric β‐trefoil protein in response to folding nucleus perturbation

Gene duplication and fusion events in protein evolution are postulated to be responsible for the common protein folds exhibiting internal rotational symmetry. Such evolutionary processes can also potentially yield regions of repetitive primary structure. Repetitive primary structure offers the potential for alternative definitions of critical regions, such as the folding nucleus (FN). In principle, more than one instance of the FN potentially enables an alternative folding pathway in the face of a subsequent deleterious mutation. We describe the targeted mutation of the carboxyl‐terminal region of the (internally located) FN of the de novo designed purely‐symmetric β‐trefoil protein Symfoil‐4P. This mutation involves wholesale replacement of a repeating trefoil‐fold motif with a “blade” motif from a β‐propeller protein, and postulated to trap that region of the Symfoil‐4P FN in a nonproductive folding intermediate. The resulting protein (termed “Bladefoil”) is shown to be cooperatively folding, but as a trimeric oligomer. The results illustrate how symmetric protein architectures have potentially diverse folding alternatives available to them, including oligomerization, when preferred pathways are perturbed.     

A simple covarion‐based approach to analyse nucleotide substitution rates

Using the ratio of nonsynonymous to synonymous nucleotide substitution rates (K_a/K_s) is a common approach for detecting positive selection. However, calculation of this ratio over a whole gene combines amino acid sites that may be under positive selection with those that are highly conserved. We introduce a new covarion‐based method to sample only the sites potentially under selective pressure. Using ancestral sequence reconstruction over a phylogenetic tree coupled with calculation of K_a/K_s ratios, positive selection is better detected by this simple covarion‐based approach than it is using a whole gene analysis or a windowing analysis. This is demonstrated on a synthetic dataset and is tested on primate leptin, which indicates a previously undetected round of positive selection in the branch leading to Gorilla gorilla.     

Structure‐guided approach for detecting large domain inserts in protein sequences as illustrated using the haloacid dehalogenase superfamily

In multi‐domain proteins, the domains typically run end‐to‐end, that is, one domain follows the C‐terminus of another domain. However, approximately 10% of multi‐domain proteins are formed by insertion of one domain sequence into that of another domain. Detecting such insertions within protein sequences is a fundamental challenge in structural biology. The haloacid dehalogenase superfamily (HADSF) serves as a challenging model system wherein a variable cap domain (～5–200 residues in length) accessorizes the ubiquitous Rossmann‐fold core domain, with variations in insertion site and topology corresponding to different classes of cap types. Herein, we describe a comprehensive computational strategy, CapPredictor, for determining large, variable domain insertions in protein sequences. Using a novel sequence‐alignment algorithm in conjunction with a structure‐guided sequence profile from 154 core‐domain‐only structures, more than 40,000 HADSF member sequences were assigned cap types. The resulting data set afforded insight into HADSF evolution. Notably, a similar distribution of cap‐type classes across different phyla was observed, indicating that all cap types existed in the last universal common ancestor. In addition, comparative analyses of the predicted cap‐type and functional assignments showed that different cap types carry out similar chemistries. Thus, while cap domains play a role in substrate recognition and chemical reactivity, cap‐type does not strictly define functional class. Through this example, we have shown that CapPredictor is an effective new tool for the study of form and function in protein families where domain insertion occurs. Proteins 2014; 82:1896–1906. © 2014 Wiley Periodicals, Inc.     

Accurate domain identification with structure‐anchored hidden Markov models,saHMMs

The ever increasing speed of DNA sequencing widens the discrepancy between the number of known gene products, and the knowledge of their function and structure. Proper annotation of protein sequences is therefore crucial if the missing information is to be deduced from sequence‐based similarity comparisons. These comparisons become exceedingly difficult as the pairwise identities drop to very low values. To improve the accuracy of domain identification, we exploit the fact that the three‐dimensional structures of domains are much more conserved than their sequences. Based on structure‐anchored multiple sequence alignments of low identity homologues we constructed 850 structure‐anchored hidden Markov models (saHMMs), each representing one domain family. Since the saHMMs are highly family specific, they can be used to assign a domain to its correct family and clearly distinguish it from domains belonging to other families, even within the same superfamily. This task is not trivial and becomes particularly difficult if the unknown domain is distantly related to the rest of the domain sequences within the family. In a search with full length protein sequences, harbouring at least one domain as defined by the structural classification of proteins database (SCOP), version 1.71, versus the saHMM database based on SCOP version 1.69, we achieve an accuracy of 99.0%. All of the few hits outside the family fall within the correct superfamily. Compared to Pfam_ls HMMs, the saHMMs obtain about 11% higher coverage. A comparison with BLAST and PSI‐BLAST demonstrates that the saHMMs have consistently fewer errors per query at a given coverage. Within our recommended E‐value range, the same is true for a comparison with SUPERFAMILY. Furthermore, we are able to annotate 232 proteins with 530 nonoverlapping domains belonging to 102 different domain families among human proteins labelled “unknown” in the NCBI protein database. Our results demonstrate that the saHMM database represents a versatile and reliable tool for identification of domains in protein sequences. With the aid of saHMMs, homology on the family level can be assigned, even for distantly related sequences. Due to the construction of the saHMMs, the hits they provide are always associated with high quality crystal structures. The saHMM database can be accessed via the FISH server at http://babel.ucmp.umu.se/fish/ . Proteins 2009. © 2008 Wiley‐Liss, Inc.     

Predicting polymorphic EST‐SSRs in silico

The public availability of large quantities of gene sequence data provides a valuable resource of the mining of Simple Sequence Repeat (SSR) molecular genetic markers for genetic analysis. These markers are inexpensive, require minimal labour to produce and can frequently be associated with functionally annotated genes. This study presents the characterization of barley EST‐SSRs and the identification of putative polymorphic SSRs from EST data. Polymorphic SSRs are distinguished from monomorphic SSRs by the representation of varying motif lengths within an alignment of sequence reads. Two measures of confidence are calculated, redundancy of a polymorphism and co‐segregation with accessions. The utility of this method is demonstrated through the discovery of 597 candidate polymorphic SSRs, from a total of 452 642 consensus expressed sequences. PCR amplification primers were designed for the identified SSRs. Ten primer pairs were validated for polymorphism in barley and for transferability across species. Analysis of the polymorphisms in relation to SSR motif, length, position and annotation is discussed.     

Invasion success: does size really matter?

The recent paper by Roy et al . (2001) presents a compelling relationship between range limit shifts, climatic fluctuations, and body size for marine bivalves in the fossil record. However, their extension of body size as a correlate for contemporary marine bivalve introductions is problematic and requires further scrutiny. Unlike their analysis of the fossil assemblage, the approach used for contemporary invasions does not adequately control for dispersal mechanism (vector) or source region. First, their analysis included mariculture species, intentionally introduced because of their large size, creating a vector-specific bias. Second, successful invaders from multiple source regions (Northern Hemisphere) were compared with potential invaders from a single source region (north-eastern Pacific), leaving both source and vector as uncontrolled variables. We present an analysis of body size for bivalve introductions from a single vector and source region, indicating no correlation between body size and invasion success when eliminating intentional introduction, source region and transport vector as confounding factors.     

Coiled‐coils: The long and short of it

Coiled‐coils are found in proteins throughout all three kingdoms of life. Coiled‐coil domains of some proteins are almost invariant in sequence and length, betraying a structural and functional role for amino acids along the entire length of the coiled‐coil. Other coiled‐coils are divergent in sequence, but conserved in length, thereby functioning as molecular spacers. In this capacity, coiled‐coil proteins influence the architecture of organelles such as centrioles and the Golgi, as well as permit the tethering of transport vesicles. Specialized coiled‐coils, such as those found in motor proteins, are capable of propagating conformational changes along their length that regulate cargo binding and motor processivity. Coiled‐coil domains have also been identified in enzymes, where they function as molecular rulers, positioning catalytic activities at fixed distances. Finally, while coiled‐coils have been extensively discussed for their potential to nucleate and scaffold large macromolecular complexes, structural evidence to substantiate this claim is relatively scarce.     

Artificial protein sequences enable recognition of vicinal and distant protein functional relationships

High divergence in protein sequences makes the detection of distant protein relationships through homology-based approaches challenging. Grouping protein sequences into families, through similarities in either sequence or 3-D structure, facilitates in the improved recognition of protein relationships. In addition, strategically designed protein-like sequences have been shown to bridge distant structural domain families by serving as artificial linkers. In this study, we have augmented a search database of known protein domain families with such designed sequences, with the intention of providing functional clues to domain families of unknown structure. When assessed using representative query sequences from each family, we obtain a success rate of 94% in protein domain families of known structure. Further, we demonstrate that the augmented search space enabled fold recognition for 582 families with no structural information available a priori. Additionally, we were able to provide reliable functional relationships for 610 orphan families. We discuss the application of our method in predicting functional roles through select examples for DUF4922, DUF5131, and DUF5085. Our approach also detects new associations between families that were previously not known to be related, as demonstrated through new sub-groups of the RNA polymerase domain among three distinct RNA viruses. Taken together, designed sequences-augmented search databases direct the detection of meaningful relationships between distant protein families. In turn, they enable fold recognition and offer reliable pointers to potential functional sites that may be probed further through direct mutagenesis studies.     

Intron/exon structure of the human gene for the muscle isozyme of glycogen phosphorylase

The intron/exon organization of the human gene for glycogen phosphorylase has been determined. The segments of the polypeptide chain that corresponds to the 19 exons of the gene are examined for relationships between the three-dimensional structure to the protein and gene structure. Only weak correlations are observed between domains of phosphorylase and exons. The nucleotide binding domains that are found in phosphorylase and other glycolytic enzymes are examined for relationships between exons of the genes and structures of the domains. When mapped to the three-dimensional structures, the intron/exon boundaries are shown to be widely distributed in this family of protein domains.     

Coiled‐coil registry shifts in the F684I mutant of Bicaudal D result in cargo‐independent activation of dynein motility

The dynein adaptor Drosophila Bicaudal D (BicD) is auto‐inhibited and activates dynein motility only after cargo is bound, but the underlying mechanism is elusive. In contrast, we show that the full‐length BicD/F684I mutant activates dynein processivity even in the absence of cargo. Our X‐ray structure of the C‐terminal domain of the BicD/F684I mutant reveals a coiled‐coil registry shift; in the N‐terminal region, the two helices of the homodimer are aligned, whereas they are vertically shifted in the wild‐type. One chain is partially disordered and this structural flexibility is confirmed by computations, which reveal that the mutant transitions back and forth between the two registries. We propose that a coiled‐coil registry shift upon cargo‐binding activates BicD for dynein recruitment. Moreover, the human homolog BicD2/F743I exhibits diminished binding of cargo adaptor Nup358, implying that a coiled‐coil registry shift may be a mechanism to modulate cargo selection for BicD2‐dependent transport pathways.     

FAIR: A server for internal sequence repeats

An Internet computing server has been developed to identify all the occurrences of the internal sequence repeats in a protein and DNA sequences. Further, an option is provided for the users to check the occurrence(s) of the resultant sequence repeats in the other sequence and structure (Protein Data Bank) databases. The databases deployed in the proposed computing engine are up-to-date and thus the users will get the latest information available in the respective databases. The server is freely accessible over the World Wide Web (WWW). AVAILABILITY: http://bioserver1.physics.iisc.ernet.in/fair/     

EST‐derived polymorphic microsatellites from cultivated strawberry (Fragaria×ananassa) are useful for diversity studies and varietal identification among Fragaria species

Microsatellite or simple sequence repeat markers derived from expressed sequence tags (ESTs) provide genetic markers within potentially functional genes, which could be very useful for breeding programs. To date, the development of microsatellite markers in the genus Fragaria has focused mainly on Fragaria vesca. However, most of the interests of breeding programs relate to specific characteristics of cultivated strawberry. Here, we describe a set of 10 EST‐derived microsatellites from Fragaria × ananassa. These markers showed high levels of polymorphism within strawberry cultivars and among different Fragaria species, indicating their potential for genetic studies not only on strawberry but also in other species within the genus.