Short Motifs Research Articles

The Hsp70-Chaperone Machines in Bacteria.

The ATP-dependent Hsp70s are evolutionary conserved molecular chaperones that constitute central hubs of the cellular protein quality surveillance network. None of the other main chaperone families (Tig, GroELS, HtpG, IbpA/B, ClpB) have been assigned with a comparable range of functions. Through a multitude of functions Hsp70s are involved in many cellular control circuits for maintaining protein homeostasis and have been recognized as key factors for cell survival. Three mechanistic properties of Hsp70s are the basis for their high versatility. First, Hsp70s bind to short degenerate sequence motifs within their client proteins. Second, Hsp70 chaperones switch in a nucleotide-controlled manner between a state of low affinity for client proteins and a state of high affinity for clients. Third, Hsp70s are targeted to their clients by a large number of cochaperones of the J-domain protein (JDP) family and the lifetime of the Hsp70-client complex is regulated by nucleotide exchange factors (NEF). In this review I will discuss advances in the understanding of the molecular mechanism of the Hsp70 chaperone machinery focusing mostly on the bacterial Hsp70 DnaK and will compare the two other prokaryotic Hsp70s HscA and HscC with DnaK.

CpGmotifs: a tool to discover DNA motifs associated to CpG methylation events

BackgroundThe investigation of molecular alterations associated with the conservation and variation of DNA methylation in eukaryotes is gaining interest in the biomedical research community. Among the different determinants of methylation stability, the DNA composition of the CpG surrounding regions has been shown to have a crucial role in the maintenance and establishment of methylation statuses. This aspect has been previously characterized in a quantitative manner by inspecting the nucleotidic composition in the region. Research in this field still lacks a qualitative perspective, linked to the identification of certain sequences (or DNA motifs) related to particular DNA methylation phenomena.ResultsHere we present a novel computational strategy based on short DNA motif discovery in order to characterize sequence patterns related to aberrant CpG methylation events. We provide our framework as a user-friendly, shiny-based application, CpGmotifs, to easily retrieve and characterize DNA patterns related to CpG methylation in the human genome. Our tool supports the functional interpretation of deregulated methylation events by predicting transcription factors binding sites (TFBS) encompassing the identified motifs.ConclusionsCpGmotifs is an open source software. Its source code is available on GitHub https://github.com/Greco-Lab/CpGmotifs and a ready-to-use docker image is provided on DockerHub at https://hub.docker.com/r/grecolab/cpgmotifs.

The impact of oncogenic mutations of the viral Src kinase on the structure and stability of the SH3 domain.

Src kinase belongs to the family of Src-related nonreceptor tyrosine kinases. Because of its physiological role in cell growth and proliferation, its activity is strictly controlled by several mechanisms. Nevertheless, in viral Src kinase (v-Src) some of these mechanisms fail, and its uncontrolled activity is responsible for the occurrence of cancer. Here, the crystal structures of three SH3-domain mutants of v-Src were determined to unveil the effects of these oncogenic mutations in this regulatory domain. Mutations in the n-Src and distal loops have a low impact on the overall structure of the domain and its capacity to form intertwined dimers. However, mutations in the RT loop compromise the stability of the domain and make the protein very prone to aggregation. Additionally, these mutations prevent the formation of intertwined dimers. The results show a synergistic effect between mutations in the RT loop and those in the n-Src and distal loops. Analysis of the structures of the v-Src SH3-domain mutants and the closed inactive conformation of cellular Src kinase (c-Src) point to a loss of the interactions that are required to establish the compact inactive form of the kinase. Nevertheless, an analysis of structures of the c-Src SH3 domain complexed with class I and II peptides points to minor changes in the interactions between the v-Src SH3 domain and these peptides. In this way, the structures reported here indicate that mutations in the RT loop might impair the kinase regulation mechanism without affecting the recognition of short proline-rich motifs in the target proteins of the kinase, thus explaining the oncogenic behaviour of the protein.

Characterization of a novel type of carbonic anhydrase that acts without metal cofactors

BackgroundCarbonic anhydrases (CAs) are universal metalloenzymes that catalyze the reversible conversion of carbon dioxide (CO2) and bicarbonate (HCO3-). They are involved in various biological processes, including pH control, respiration, and photosynthesis. To date, eight evolutionarily unrelated classes of CA families (α, β, γ, δ, ζ, η, θ, and ι) have been identified. All are characterized by an active site accommodating the binding of a metal cofactor, which is assumed to play a central role in catalysis. This feature is thought to be the result of convergent evolution.ResultsHere, we report that a previously uncharacterized protein group, named “COG4337,” constitutes metal-independent CAs from the newly discovered ι-class. Genes coding for COG4337 proteins are found in various bacteria and photosynthetic eukaryotic algae. Biochemical assays demonstrated that recombinant COG4337 proteins from a cyanobacterium (Anabaena sp. PCC7120) and a chlorarachniophyte alga (Bigelowiella natans) accelerated CO2 hydration. Unexpectedly, these proteins exhibited their activity under metal-free conditions. Based on X-ray crystallography and point mutation analysis, we identified a metal-free active site within the cone-shaped α+β barrel structure. Furthermore, subcellular localization experiments revealed that COG4337 proteins are targeted into plastids and mitochondria of B. natans, implicating their involvement in CO2 metabolism in these organelles.ConclusionsCOG4337 proteins shared a short sequence motif and overall structure with ι-class CAs, whereas they were characterized by metal independence, unlike any known CAs. Therefore, COG4337 proteins could be treated as a variant type of ι-class CAs. Our findings suggested that this novel type of ι-CAs can function even in metal-poor environments (e.g., the open ocean) without competition with other metalloproteins for trace metals. Considering the widespread prevalence of ι-CAs across microalgae, this class of CAs may play a role in the global carbon cycle.

Therapeutic targeting of RNA-binding protein by RNA-PROTAC

Therapeutic targeting of RNA-binding protein by RNA-PROTAC

Phosphorylation of the WH2 domain in yeast Las17/WASP regulates G-actin binding and protein function during endocytosis

Actin nucleation is the key rate limiting step in the process of actin polymerization, and tight regulation of this process is critical to ensure actin filaments form only at specific times and at defined regions of the cell. WH2 domains are short sequence motifs found in many different actin binding proteins including WASP family proteins which regulate the actin nucleating complex Arp2/3. In this study we reveal a phosphorylation site, Serine 554, within the WH2 domain of the yeast WASP homologue Las17. Both phosphorylation and a phospho-mimetic mutation reduce actin monomer binding affinity while an alanine mutation, generated to mimic the non-phosphorylated state, increases actin binding affinity. The effect of these mutations on the Las17-dependent process of endocytosis in vivo was analysed and leads us to propose that switching of Las17 phosphorylation states may allow progression through distinct phases of endocytosis from site assembly through to the final scission stage. While the study is focused on Las17, the sole WASP family protein in yeast, our results have broad implications for our understanding of how a key residue in this conserved motif can underpin the many different actin regulatory roles with which WH2 domains have been associated.

Identification and evaluation of Calotropis procera phytocompounds against novel protein PE_PGRS 16 from Mycobacterium tuberculosis H37Rv

The authors are currently dealing with the highest incidence of tuberculosis, where every third person in the world is experiencing the latent condition of the disease. The affected ratio is not limited to any age group, and therefore, the development of new drug or vaccine is the need of the hour. The use of medicinal plants in curing disease is a novel and significant area of interest. The PE_PGRS 16 has putative aspartic proteinase domain, which reveals the characteristic of pepsin-fold and is recognized as a surface antigen; therefore, it would be an important target for drug design. The structure of PE_PGRS 16 was obtained from PDB databank and the phytocompounds of Calotropis procera were obtained from gas chromatography–mass spectrometry analysis. PyRx tool and AutoDock Vina were used to prepared dock file and docking analysis, respectively. The compounds were selected based on docking score, and selected compounds were further checked by absorption, distribution, metabolism, and excretion (ADME) properties to conclude a list of safe and effective drugs. Visualization and analysis of interaction was done by Discovery Studio Visualizer. Screened phytocompounds against the PE_PGRS 16 accurately fitted within the active sites. The structural analysis suggested the binding to residues Asp46, Thr47, and Gly48 short sequence motif D(T/S)G 1, Asp199, Ser200, and Gly201 D(T/S)G 2 with the compound. These residues are important for protein activity, and therefore, drug binding at these residues may hamper protein’s activity. l-rhamnose and ferulic acid closely interacted with active site residue of PE_PGRS 16, and therefore, it is hypothesized that these compounds are the putative target of the protein activity, which enhance bacterial pathogenesis and survival.

Bora phosphorylation substitutes in trans for T-loop phosphorylation in Aurora A to promote mitotic entry

Polo-like kinase 1 (Plk1) is instrumental for mitotic entry and progression. Plk1 is activated by phosphorylation on a conserved residue Thr210 in its activation segment by the Aurora A kinase (AURKA), a reaction that critically requires the co-factor Bora phosphorylated by a CyclinA/B-Cdk1 kinase. Here we show that phospho-Bora is a direct activator of AURKA kinase activity. We localize the key determinants of phospho-Bora function to a 100 amino acid region encompassing two short Tpx2-like motifs and a phosphoSerine-Proline motif at Serine 112, through which Bora binds AURKA. The latter substitutes in trans for the Thr288 phospho-regulatory site of AURKA, which is essential for an active conformation of the kinase domain. We demonstrate the importance of these determinants for Bora function in mitotic entry both in Xenopus egg extracts and in human cells. Our findings unveil the activation mechanism of AURKA that is critical for mitotic entry.

STREME: accurate and versatile sequence motif discovery.

Sequence motif discovery algorithms can identify novel sequence patterns that perform biological functions in DNA, RNA and protein sequences-for example, the binding site motifs of DNA- and RNA-binding proteins. The STREME algorithm presented here advances the state-of-the-art in ab initio motif discovery in terms of both accuracy and versatility. Using in vivo DNA (ChIP-seq) and RNA (CLIP-seq) data, and validating motifs with reference motifs derived from in vitro data, we show that STREME is more accurate, sensitive and thorough than several widely used algorithms (DREME, HOMER, MEME, Peak-motifs) and two other representative algorithms (ProSampler and Weeder). STREME's capabilities include the ability to find motifs in datasets with hundreds of thousands of sequences, to find both short and long motifs (from 3 to 30 positions), to perform differential motif discovery in pairs of sequence datasets, and to find motifs in sequences over virtually any alphabet (DNA, RNA, protein and user-defined alphabets). Unlike most motif discovery algorithms, STREME reports a useful estimate of the statistical significance of each motif it discovers. STREME is easy to use individually via its web server or via the command line, and is completely integrated with the widely used MEME Suite of sequence analysis tools. The name STREME stands for 'Simple, Thorough, Rapid, Enriched Motif Elicitation'. The STREME web server and source code are provided freely for non-commercial use at http://meme-suite.org. Supplementary data are available at Bioinformatics online.

Multiple, short protein binding motifs in ORC1 and CDC6 control the initiation of DNA replication

The initiation of DNA replication involves cell cycle-dependent assembly and disassembly of protein complexes, including the origin recognition complex (ORC) and CDC6 AAA+ ATPases. We report that multiple short linear protein motifs (SLiMs) within intrinsically disordered regions (IDRs) in ORC1 and CDC6 mediate cyclin-CDK-dependent and independent protein-protein interactions, conditional on the cell cycle phase. A domain within the ORC1 IDR is required for interaction between the ORC1 and CDC6 AAA+ domains in G1, whereas the same domain prevents CDC6-ORC1 interaction during mitosis. Then, during late G1, this domain facilitates ORC1 destruction by a SKP2-cyclin A-CDK2-dependent mechanism. During G1, the CDC6 Cy motif cooperates with cyclin E-CDK2 to promote ORC1-CDC6 interactions. The CDC6 IDR regulates self-interaction by ORC1, thereby controlling ORC1 protein levels. Protein phosphatase 1 binds directly to a SLiM in the ORC1 IDR, causing ORC1 de-phosphorylation upon mitotic exit, increasing ORC1 protein, and promoting pre-RC assembly.

Archaeal Connectase is a specific and efficient protein ligase related to proteasome β subunits

Sequence-specific protein ligations are widely used to produce customized proteins "on demand." Such chimeric, immobilized, fluorophore-conjugated or segmentally labeled proteins are generated using a range of chemical, (split) intein, split domain, or enzymatic methods. Where short ligation motifs and good chemoselectivity are required, ligase enzymes are often chosen, although they have a number of disadvantages, for example poor catalytic efficiency, low substrate specificity, and side reactions. Here, we describe a sequence-specific protein ligase with more favorable characteristics. This ligase, Connectase, is a monomeric homolog of 20S proteasome subunits in methanogenic archaea. In pulldown experiments with Methanosarcina mazei cell extract, we identify a physiological substrate in methyltransferase A (MtrA), a key enzyme of archaeal methanogenesis. Using microscale thermophoresis and X-ray crystallography, we show that only a short sequence of about 20 residues derived from MtrA and containing a highly conserved KDPGA motif is required for this high-affinity interaction. Finally, in quantitative activity assays, we demonstrate that this recognition tag can be repurposed to allow the ligation of two unrelated proteins. Connectase catalyzes such ligations at substantially higher rates, with higher yields, but without detectable side reactions when compared with a reference enzyme. It thus presents an attractive tool for the development of new methods, for example in the preparation of selectively labeled proteins for NMR, the covalent and geometrically defined attachment of proteins on surfaces for cryo-electron microscopy, or the generation of multispecific antibodies.

NMR structure and dynamics of Q4DY78, a conserved kinetoplasid-specific protein from Trypanosoma cruzi

The 106-residue protein Q4DY78 (UniProt accession number) from Trypanosoma cruzi is highly conserved in the related kinetoplastid pathogens Trypanosoma brucei and Leishmania major. Given the essentiality of its orthologue in T. brucei, the high sequence conservation with other trypanosomatid proteins, and the low sequence similarity with mammalian proteins, Q4DY78 is an attractive protein for structural characterization. Here, we solved the structure of Q4DY78 by solution NMR and evaluated its backbone dynamics. Q4DY78 is composed of five α -helices and a small, two-stranded antiparallel β-sheet. The backbone RMSD is 0.22 ± 0.05 Å for the representative ensemble of the 20 lowest-energy structures. Q4DY78 is overall rigid, except for N-terminal residues (V8 to I10), residues at loop 4 (K57 to G65) and residues at the C-terminus (F89 to F112). Q4DY78 has a short motif FPCAP that could potentially mediate interactions with the host cytoskeleton via interaction with EVH1 (Drosophila Enabled (Ena)/Vasodilator-stimulated phosphoprotein (VASP) homology 1) domains. Albeit Q4DY78 lacks calcium-binding motifs, its fold resembles that of eukaryotic calcium-binding proteins such as calcitracin, calmodulin, and polcacin Bet V4. We characterized this novel protein with a calcium binding fold without the capacity to bind calcium.

Plxdc family members are novel receptors for the rhesus monkey rhadinovirus (RRV).

The rhesus monkey rhadinovirus (RRV), a γ2-herpesvirus of rhesus macaques, shares many biological features with the human pathogenic Kaposi’s sarcoma-associated herpesvirus (KSHV). Both viruses, as well as the more distantly related Epstein-Barr virus, engage cellular receptors from the Eph family of receptor tyrosine kinases (Ephs). However, the importance of the Eph interaction for RRV entry varies between cell types suggesting the existence of Eph-independent entry pathways. We therefore aimed to identify additional cellular receptors for RRV by affinity enrichment and mass spectrometry. We identified an additional receptor family, the Plexin domain containing proteins 1 and 2 (Plxdc1/2) that bind the RRV gH/gL glycoprotein complex. Preincubation of RRV with soluble Plxdc2 decoy receptor reduced infection by ~60%, while overexpression of Plxdc1 and 2 dramatically enhanced RRV susceptibility and cell-cell fusion of otherwise marginally permissive Raji cells. While the Plxdc2 interaction is conserved between two RRV strains, 26–95 and 17577, Plxdc1 specifically interacts with RRV 26–95 gH. The Plxdc interaction is mediated by a short motif at the N-terminus of RRV gH that is partially conserved between isolate 26–95 and isolate 17577, but absent in KSHV gH. Mutation of this motif abrogated the interaction with Plxdc1/2 and reduced RRV infection in a cell type-specific manner. Taken together, our findings characterize Plxdc1/2 as novel interaction partners and entry receptors for RRV and support the concept of the N-terminal domain of the gammaherpesviral gH/gL complex as a multifunctional receptor-binding domain. Further, Plxdc1/2 usage defines an important biological difference between KSHV and RRV.

Avoided motifs: short amino acid strings missing from protein datasets.

According to the amino acid composition of natural proteins, it could be expected that all possible sequences of three or four amino acids will occur at least once in large protein datasets purely by chance. However, in some species or cellular context, specific short amino acid motifs are missing due to unknown reasons. We describe these as Avoided Motifs, short amino acid combinations missing from biological sequences. Here we identify 209 human and 154 bacterial Avoided Motifs of length four amino acids, and discuss their possible functionality according to their presence in other species. Furthermore, we determine two Avoided Motifs of length three amino acids in human proteins specifically located in the cytoplasm, and two more in secreted proteins. Our results support the hypothesis that the characterization of Avoided Motifs in particular contexts can provide us with information about functional motifs, pointing to a new approach in the use of molecular sequences for the discovery of protein function.

Evolution and Diversity of Semaphorins and Plexins in Choanoflagellates.

Semaphorins and plexins are cell surface ligand/receptor proteins that affect cytoskeletal dynamics in metazoan cells. Interestingly, they are also present in Choanoflagellata, a class of unicellular heterotrophic flagellates that forms the phylogenetic sister group to Metazoa. Several members of choanoflagellates are capable of forming transient colonies, whereas others reside solitary inside exoskeletons; their molecular diversity is only beginning to emerge. Here, we surveyed genomics data from 22 choanoflagellate species and detected semaphorin/plexin pairs in 16 species. Choanoflagellate semaphorins (Sema-FN1) contain several domain features distinct from metazoan semaphorins, including an N-terminal Reeler domain that may facilitate dimer stabilization, an array of fibronectin type III domains, a variable serine/threonine-rich domain that is a potential site for O-linked glycosylation, and a SEA domain that can undergo autoproteolysis. In contrast, choanoflagellate plexins (Plexin-1) harbor a domain arrangement that is largely identical to metazoan plexins. Both Sema-FN1 and Plexin-1 also contain a short homologous motif near the C-terminus, likely associated with a shared function. Three-dimensional molecular models revealed a highly conserved structural architecture of choanoflagellate Plexin-1 as compared to metazoan plexins, including similar predicted conformational changes in a segment that is involved in the activation of the intracellular Ras-GAP domain. The absence of semaphorins and plexins in several choanoflagellate species did not appear to correlate with unicellular versus colonial lifestyle or ecological factors such as fresh versus salt water environment. Together, our findings support a conserved mechanism of semaphorin/plexin proteins in regulating cytoskeletal dynamics in unicellular and multicellular organisms.

Receptor-like role for PQLC2 amino acid transporter in the lysosomal sensing of cationic amino acids

PQLC2, a lysosomal cationic amino acid transporter, also serves as a sensor that responds to scarcity of its substrates by recruiting a protein complex composed of C9orf72, SMCR8, and WDR41 to the surface of lysosomes. This protein complex controls multiple aspects of lysosome function. Although it is known that this response to changes in cationic amino acid availability depends on an interaction between PQLC2 and WDR41, the underlying mechanism for the regulated interaction is not known. In this study, we present evidence that the WDR41-PQLC2 interaction is mediated by a short peptide motif in a flexible loop that extends from the WDR41 β-propeller and inserts into a cavity presented by the inward-facing conformation of PQLC2. The data support a transceptor model wherein conformational changes in PQLC2 related to substrate transport regulate the availability of the WDR41-binding site on PQLC2 and mediate recruitment of the WDR41-SMCR8-C9orf72 complex to the surface of lysosomes.

Characterization of the Structural Features that Mediate Phase Separation of Intrinsically Disordered Low-Complexity Domains

Recently, much insight has been gained regarding the sequence features of intrinsically disordered low complexity domains that drive the formation of biomolecular condensates. However, there is still controversy regarding what structural features drive condensate formation. One school of thought posits that transient interactions are formed between sticky side chains in an otherwise disordered chain, even inside of dense phases. The other side argues that interactions between short motifs that form cross-β structure drives phase separation.

Electrical DNA Sequence Mapping Using Oligodeoxynucleotide Labels and Nanopores.

Identifying DNA species is crucial for diagnostics. For DNA identification, single-molecule DNA sequence mapping is an alternative to DNA sequencing toward fast point-of-care testing, which traditionally relies on targeting and labeling DNA sequences with fluorescent labels and readout using optical imaging methods. A nanopore is a promising sensor as a complement to optical mapping with advantages of electric measurement suitable for portable devices and potential for high resolution. Here, we demonstrate a high-resolution nanopore-based DNA sequence mapping by labeling specific short sequence motifs with oligodeoxynucleotides (ODNs) using DNA methyltransferase (MTase) and detecting them using nanopores. We successfully detected ODNs down to the size of 11 nucleotides without introducing extra reporters and resolved neighboring sites with a distance of 141 bp (∼48 nm) on a single DNA molecule. To accurately locate the sequence motif positions on DNA, a nanopore data analysis method is proposed by considering DNA velocity change through nanopores and using ensemble statistics to translate the time-dependent signals to the location information. Our platform enables high-resolution detection of small labels on DNA and high-accuracy localization of them for DNA species identification in an all-electrical format. The method presents an alternative to optical techniques relying on fluorescent labels and is promising for miniature-scale integration for diagnostic applications.

Deep neural networks identify sequence context features predictive of transcription factor binding.

Transcription factors (TFs) bind DNA by recognizing specific sequence motifs, typically of length 6–12bp. A motif can occur many thousands of times in the human genome, but only a subset of those sites are actually bound. Here we present a machine learning framework leveraging existing convolutional neural network architectures and model interpretation techniques to identify and interpret sequence context features most important for predicting whether a particular motif instance will be bound. We apply our framework to predict binding at motifs for 38 TFs in a lymphoblastoid cell line, score the importance of context sequences at base-pair resolution, and characterize context features most predictive of binding. We find that the choice of training data heavily influences classification accuracy and the relative importance of features such as open chromatin. Overall, our framework enables novel insights into features predictive of TF binding and is likely to inform future deep learning applications to interpret non-coding genetic variants.

MicroProteins: Expanding functions and novel modes of regulation

MicroProteins: Expanding functions and novel modes of regulation