The BLAST programs are widely used tools for searching protein and DNA databases for sequence similarities. For protein comparisons, a variety of definitional, algorithmic and statistical refinements described here permits the execution time of the BLAST programs to be decreased substantially while enhancing their sensitivity to weak similarities. A new criterion for triggering the extension of word hits, combined with a new heuristic for generating gapped alignments, yields a gapped BLAST program that runs at approximately three times the speed of the original. In addition, a method is introduced for automatically combining statistically significant alignments produced by BLAST into a position-specific score matrix, and searching the database using this matrix. The resulting Position-Specific Iterated BLAST (PSIBLAST) program runs at approximately the same speed per iteration as gapped BLAST, but in many cases is much more sensitive to weak but biologically relevant sequence similarities. PSI-BLAST is used to uncover several new and interesting members of the BRCT superfamily.

/pdf/gapped-blast-and-psi-blast-a-new-generation-of-protein-2ybl67x5yx.pdf

Gapped BLAST and PSI-BLAST: a new generation of protein database search programs.

The sensitivity of the commonly used progressive multiple sequence alignment method has been greatly improved for the alignment of divergent protein sequences. Firstly, individual weights are assigned to each sequence in a partial alignment in order to down-weight near-duplicate sequences and up-weight the most divergent ones. Secondly, amino acid substitution matrices are varied at different alignment stages according to the divergence of the sequences to be aligned. Thirdly, residue-specific gap penalties and locally reduced gap penalties in hydrophilic regions encourage new gaps in potential loop regions rather than regular secondary structure. Fourthly, positions in early alignments where gaps have been opened receive locally reduced gap penalties to encourage the opening up of new gaps at these positions. These modifications are incorporated into a new program, CLUSTAL W which is freely available.

/pdf/clustal-w-improving-the-sensitivity-of-progressive-multiple-2na2s9sq2h.pdf

Clustal w: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice

CLUSTAL X is a new windows interface for the widely-used progressive multiple sequence alignment program CLUSTAL W. The new system is easy to use, providing an integrated system for performing multiple sequence and profile alignments and analysing the results. CLUSTAL X displays the sequence alignment in a window on the screen. A versatile sequence colouring scheme allows the user to highlight conserved features in the alignment. Pull-down menus provide all the options required for traditional multiple sequence and profile alignment. New features include: the ability to cut-and-paste sequences to change the order of the alignment, selection of a subset of the sequences to be realigned, and selection of a sub-range of the alignment to be realigned and inserted back into the original alignment. Alignment quality analysis can be performed and low-scoring segments or exceptional residues can be highlighted. Quality analysis and realignment of selected residue ranges provide the user with a powerful tool to improve and refine difficult alignments and to trap errors in input sequences. CLUSTAL X has been compiled on SUN Solaris, IRIX5.3 on Silicon Graphics, Digital UNIX on DECstations, Microsoft Windows (32 bit) for PCs, Linux ELF for x86 PCs, and Macintosh PowerMac.

/pdf/the-clustal-x-windows-interface-flexible-strategies-for-3qez0eyvqt.pdf

The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools.

We describe MUSCLE, a new computer program for creating multiple alignments of protein sequences. Elements of the algorithm include fast distance estimation using kmer counting, progressive alignment using a new profile function we call the logexpectation score, and refinement using treedependent restricted partitioning. The speed and accuracy of MUSCLE are compared with T-Coffee, MAFFT and CLUSTALW on four test sets of reference alignments: BAliBASE, SABmark, SMART and a new benchmark, PREFAB. MUSCLE achieves the highest, or joint highest, rank in accuracy on each of these sets. Without refinement, MUSCLE achieves average accuracy statistically indistinguishable from T-Coffee and MAFFT, and is the fastest of the tested methods for large numbers of sequences, aligning 5000 sequences of average length 350 in 7 min on a current desktop computer. The MUSCLE program, source code and PREFAB test data are freely available at http://www.drive5. com/muscle.

MUSCLE: multiple sequence alignment with high accuracy and high throughput

The design, implementation, and capabilities of an extensible visualization system, UCSF Chimera, are discussed. Chimera is segmented into a core that provides basic services and visualization, and extensions that provide most higher level functionality. This architecture ensures that the extension mechanism satisfies the demands of outside developers who wish to incorporate new features. Two unusual extensions are presented: Multiscale, which adds the ability to visualize large-scale molecular assemblies such as viral coats, and Collaboratory, which allows researchers to share a Chimera session interactively despite being at separate locales. Other extensions include Multalign Viewer, for showing multiple sequence alignments and associated structures; ViewDock, for screening docked ligand orientations; Movie, for replaying molecular dynamics trajectories; and Volume Viewer, for display and analysis of volumetric data. A discussion of the usage of Chimera in real-world situations is given, along with anticipated future directions. Chimera includes full user documentation, is free to academic and nonprofit users, and is available for Microsoft Windows, Linux, Apple Mac OS X, SGI IRIX, and HP Tru64 Unix from http://www.cgl.ucsf.edu/chimera/.

/pdf/ucsf-chimera-a-visualization-system-for-exploratory-research-59xu54fhxg.pdf

UCSF Chimera--a visualization system for exploratory research and analysis.

The primary cell surface receptor for SARS-CoV-2 is the angiotensin-converting enzyme 2 (ACE2). Recently it has been noticed that the viral Spike protein has an RGD motif, suggesting that cell surface integrins may be co-receptors. We examined the sequences of ACE2 and integrins with the Eukaryotic Linear Motif resource, ELM, and were presented with candidate short linear motifs (SLiMs) in their short, unstructured, cytosolic tails with potential roles in endocytosis, membrane dynamics, autophagy, cytoskeleton and cell signalling. These SLiM candidates are highly conserved in vertebrates. They suggest potential interactions with the AP2 mu2 subunit as well as I-BAR, LC3, PDZ, PTB and SH2 domains found in signalling and regulatory proteins present in epithelial lung cells. Several motifs overlap in the tail sequences, suggesting that they may act as molecular switches, often involving tyrosine phosphorylation status. Candidate LIR motifs are present in the tails of ACE2 and integrin beta3, suggesting that these proteins can directly recruit autophagy components. We also noticed that the extracellular part of ACE2 has a conserved MIDAS structural motif, which are commonly used by beta integrins for ligand binding, potentially supporting the proposal that integrins and ACE2 share common ligands. The findings presented here identify several molecular links and testable hypotheses that might help uncover the mechanisms of SARS-CoV-2 attachment, entry and replication, and strengthen the possibility that it might be possible to develop host-directed therapies to dampen the efficiency of viral entry and hamper disease progression. The strong sequence conservation means that these putative SLiMs are good candidates: Nevertheless, SLiMs must always be validated by experimentation before they can be stated to be functional.

Short linear motif candidates in the cell entry system used by SARS-CoV-2 and their potential therapeutic implications

In the HLH-LZ protein family, the helix-loop-helix DNA-binding dimerization domain is followed in the sequence by a leucine zipper motif. The precise function of this second dimerization domain is still unclear, since the HLH motif of a subset of this family has been shown to be necessary and sufficient for dimerization. However, deletion and mutagenesis studies of the leucine zipper in various HLH-LZ proteins have shown a clear influence of this motif on homo- and heterodimerization. In this paper, we present a structural characterization of synthetic peptides encompassing the leucine zipper sequences of c-Myc and TFEB, using circular dichroism, analytical ultracentrifugation, and nuclear magnetic resonance. We show that the different ability of the synthetic leucine zippers of c-Myc and TFEB to homodimerize at neutral pH reflects the different dimerization properties reported for the entire proteins. The TFEB protein is known to form homodimers. c-Myc, on the other hand, does not homodimerize in vivo, but is mostly found in heterodimeric complexes with Max, another protein of the HLH-LZ family. Accordingly, our results show that the TFEB peptide homodimerizes at neutral pH whereas the Myc peptide dimerizes to a comparable amount only at acidic pH and high ionic strength. Both synthetic peptides are far less stable than leucine zippers of the b-ZIP family. The relative stability of the two leucine zippers and the factors which stabilize the dimer formation are discussed.

The dimerization stability of the HLH-LZ transcription protein family is modulated by the leucine zippers: a CD and NMR study of TFEB and c-Myc.

Motivation: KEN-box-mediated target selection is one of the mechanisms used in the proteasomal destruction of mitotic cell cycle proteins via the APC/C complex. While annotating the Eukaryotic Linear Motif resource (ELM, http://elm.eu.org/), we found that KEN motifs were significantly enriched in human protein entries with cell cycle keywords in the UniProt/Swiss-Prot database—implying that KEN-boxes might be more common than reported.

Results: Matches to short linear motifs in protein database searches are not, per se, significant. KEN-box enrichment with cell cycle Gene Ontology terms suggests that collectively these motifs are functional but does not prove that any given instance is so. Candidates were surveyed for native disorder prediction using GlobPlot and IUPred and for motif conservation in homologues. Among >25 strong new candidates, the most notable are human HIPK2, CHFR, CDC27, Dab2, Upf2, kinesin Eg5, DNA Topoisomerase 1 and yeast Cdc5 and Swi5. A similar number of weaker candidates were present. These proteins have yet to be tested for APC/C targeted destruction, providing potential new avenues of research.

Contact: toby.gibson@embl.de

Supplementary information: Tables of KEN-box candidates and keyword/conservation significance assessments are available as supplementary data at Bioinformatics online.

Discovery of candidate KEN-box motifs using Cell Cycle keyword enrichment combined with native disorder prediction and motif conservation

https://febs.onlinelibrary.wiley.com/doi/epdf/10.1016/j.febslet.2012.04.048

RACK1 research – ships passing in the night?

BACKGROUND: Linear motifs are short modules of protein sequences that play a crucial role in mediating and regulating many protein-protein interactions. The function of linear motifs strongly depends on the context, e.g. functional instances mainly occur inside flexible regions that are accessible for interaction. Sometimes linear motifs appear as isolated islands of conservation in multiple sequence alignments. However, they also occur in larger blocks of sequence conservation, suggesting an active role for the neighbouring amino acids. RESULTS: The evolution of regions flanking 116 functional linear motif instances was studied. The conservation of the amino acid sequence and order/disorder tendency of those regions was related to presence/absence of the instance. For the majority of the analysed instances, the pairs of sequences conserving the linear motif were also observed to maintain a similar local structural tendency and/or to have higher local sequence conservation when compared to pairs of sequences where one is missing the linear motif. Furthermore, those instances have a higher chance to co-evolve with the neighbouring residues in comparison to the distant ones. Those findings are supported by examples where the regulation of the linear motif-mediated interaction has been shown to depend on the modifications (e.g. phosphorylation) at neighbouring positions or is thought to benefit from the binding versatility of disordered regions. CONCLUSION: The results suggest that flanking regions are relevant for linear motif-mediated interactions, both at the structural and sequence level. More interestingly, they indicate that the prediction of linear motif instances can be enriched with contextual information by performing a sequence analysis similar to the one presented here. This can facilitate the understanding of the role of these predicted instances in determining the protein function inside the broader context of the cellular network where they arise.

/pdf/evidence-for-the-concerted-evolution-between-short-linear-61iwfa2h3b.pdf

Toby J. Gibson

Papers

Short linear motif candidates in the cell entry system used by SARS-CoV-2 and their potential therapeutic implications

The dimerization stability of the HLH-LZ transcription protein family is modulated by the leucine zippers: a CD and NMR study of TFEB and c-Myc.

Discovery of candidate KEN-box motifs using Cell Cycle keyword enrichment combined with native disorder prediction and motif conservation

RACK1 research – ships passing in the night?

Evidence for the concerted evolution between short linear protein motifs and their flanking regions.