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BackgroundAbelson tyrosine kinase (Abl) plays a key role in axon guidance in linking guidance receptors to actin dynamics. The long C-terminal domain (CTD) of Drosophila Abl is important for this role, and previous work identified the ‘first quarter’ (1Q) of the CTD as essential. Here, we link the physical interactions of 1Q binding partners to Abl’s function in axon guidance.MethodsProtein binding partners of 1Q were identified by GST pulldown and mass spectrometry, and validated using axon guidance assays in the embryonic nerve cord and motoneurons. The role of 1Q was assessed genetically, utilizing a battery ofAbltransgenes in combination with mutation or overexpression of the genes of pulled down proteins, and their partners in actin dynamics. The set ofAbltransgenes had the following regions deleted: all of 1Q, each half of 1Q (‘eighths’, 1E and 2E) or a PxxP motif in 2E, which may bind SH3 domains.ResultsGST pulldown identified Hem and Sra-1 as binding partners of 1Q, and our genetic analyses show that both proteins function with Abl in axon guidance, with Sra-1 likely interacting with 1Q. As Hem and Sra-1 are part of the actin-polymerizing WAVE regulatory complex (WRC), we extended our analyses to Abi and Trio, which interact with Abl and WRC members. Overall, the 1Q region (and especially 2E and its PxxP motif) are important for Abl’s ability to work with WRC in axon guidance. These areas are also important for Abl’s ability to function with the actin regulator Enabled. In comparison, 1E contributes to Abl function with the WRC at the midline, but less so with Enabled.ConclusionsThe 1Q region, and especially the 2E region with its PxxP motif, links Abl with the WRC, its regulators Trio and Abi, and the actin regulator Ena. Removing 1E has specific effects suggesting it may help modulate Abl’s interaction with the WRC or Ena. Thus, the 1Q region of Abl plays a key role in regulating actin dynamics during axon guidance.

The Escherichia coli single‐strand DNA binding protein (SSB) is essential to viability where it functions to regulate SSB interactome function. Here it binds to single‐stranded DNA and to target proteins that comprise the interactome. The region of SSB that links these two essential protein functions is the intrinsically disordered linker. Key to linker function is the presence of three, conserved PXXP motifs that mediate binding to oligosaccharide‐oligonucleotide binding folds (OB‐fold) present in SSB and its interactome partners. Not surprisingly, partner OB‐fold deletions eliminate SSB binding. Furthermore, single point mutations in either the PXXP motifs or, in the RecG OB‐fold, obliterate SSB binding. The data also demonstrate that, and in contrast to the view currently held in the field, the C‐terminal acidic tip of SSB is not required for interactome partner binding. Instead, we propose the tip has two roles. First, and consistent with the proposal of Dixon, to regulate the structure of the C‐terminal domain in a biologically active conformation that prevents linkers from binding to SSB OB‐folds until this interaction is required. Second, as a secondary binding domain. Finally, as OB‐folds are present in SSB and many of its partners, we present the SSB interactome as the first family of OB‐fold genome guardians identified in prokaryotes.

Grass carp reovirus (GCRV) seriously affects the grass carp farming, as the causative agent of grass carp hemorrhagic disease. In this study, we found the GCRV 096 NS38 protein contained one conservative PXXP motif similar to σNS of avian reovirus, with very low similarity rates with its homologous GCRV proteins of different genotypes. Furthermore, the soluble GCRV 096 NS38 protein was expressed in Escherichia coli, antiserum was raised and characterised. Addition of purified NS38 protein to culture media of Ctenopharyngodon idella kidney (CIK) cells clearly inhibited replication of GCRV 096 (genotype I); however, replication of GCRV GD108 (genotype Ш) was not significantly affected in CIK cells under the same conditions. It was not possible to induce cross-protection among GCRVs of different genotypes. GCRV multi-genotype should be considered and the NS38 protein was a promising candidate for developing therapies against grass carp hemorrhagic disease. Additionally, bioinformatics analysis suggested that the NS38 protein may be involved in viral genome replication. Keywords: Expression, Functional analysis, Grass carp reovirus 096, NS38 protein, Replication

PMAP-23, a cathelicidin-derived host defense peptide, does not cause severe membrane permeabilization, but exerts strong and broad-spectrum bactericidal activity. We have previously shown that it forms an amphipathic α-helical structure with a central hinge induced by the PXXP motif, which is implicated in the interaction of PMAP-23 with negatively charged bacterial membranes. Here, we studied the potential roles of the PXXP motif in PMAP-23 translocation across the lipid bilayer by replacing Pro residues with either α-helix former Ala (PMAP-PA) or α-helix breaker Gly (PMAP-PG). Although both PMAP-PA and PMAP-PG led to effective membrane depolarization and permeabilization, they showed less antimicrobial activity than wild-type PMAP-23. Interestingly, we observed that PMAP-23 crossed lipid bilayers much more efficiently than its Pro-substituted derivatives. The fact that the Gly-induced hinge was unable to replace the PXXP motif in PMAP-23 translocation suggests that the PXXP motif has unique structural properties other than the central hinge. Surface plasmon resonance sensorgrams showed that the running buffer almost entirely dissociated PMAP-23 from the membrane surface, while its Pro-substituted derivatives remained significantly bound to the membrane. In addition, kinetic analysis of the sensorgrams revealed that the central PXXP motif allows PMAP-23 to rapidly translocate at the interface between the hydrophilic and hydrophobic phases. Taken together, we propose that the structural and kinetic understanding of the PXXP motif in peptide translocation could greatly aid the development of novel antimicrobial peptides with intracellular targets by promoting peptide entry into bacterial cells.

Bag3 has been implicated in a wide variety of physiological processes from autophagy to aggresome formation and from cell transformation to survival. We argue that involvement of Bag3 in many of these processes is due to its distinct function in cell signaling. The structure of Bag3 suggests that it can serve as a scaffold that links molecular chaperones Hsp70 and small Hsps with components of a variety of signaling pathways. Major protein-protein interaction motifs of Bag3 that recruit components of signaling pathways are WW domain and PXXP motif that interacts with SH3-domain proteins. Furthermore, Hsp70-Bag3 appears to be a sensor of abnormal polypeptides during the proteotoxic stress. It also serves as a sensor of a mechanical force during mechanotransduction. Common feature of these and probably certain other sensory mechanisms is that they represent responses to specific kinds of abnormal proteins, i.e. unfolded filamin A in case of mechanotransduction or stalled translating polypeptides in case of sensing proteasome inhibition. Overall Hsp70-Bag3 module represents a novel signaling node that responds to multiple stimuli and controls multiple physiological processes.

Motivation More than half of the human proteome contains the proline-rich motif, PxxP. This motif has a high propensity for adopting a left-handed polyproline II (PPII) helix and can potentially bind SH3 domains. SH3 domains are generally grouped into two classes, based on whether the PPII binds in a positive (N-to-C terminal) or negative (C-to-N terminal) orientation. Since the discovery of this structural motif, over six decades ago, a systematic understanding of its binding remains poor and the consensus amino acid sequence that binds SH3 domains is still ill defined. Results Here, we show that the PPII interaction with SH3 domains is governed by the helix backbone and its prolines, and their rotation angle around the PPII helical axis. Based on a geometric analysis of 131 experimentally solved SH3 domains in complex with PPIIs, we observed a rotary translation along the helical screw axis, and separated them by 120° into three categories we name α (0–120°), β (120–240°) and γ (240–360°). Furthermore, we found that PPII helices are distinguished by a shifting PxxP motif preceded by positively charged residues which act as a structural reading frame and dictates the organization of SH3 domains; however, there is no one single consensus motif for all classified PPIIs. Our results demonstrate a remarkable apparatus of a lock with a rotating and translating key with no known equivalent machinery in molecular biology. We anticipate our model to be a starting point for deciphering the PPII code, which can unlock an exponential growth in our understanding of the relationship between protein structure and function. Availability and implementation We have implemented the proposed methods in the R software environment and in an R package freely available at https://github.com/Grantlab/bio3d. Supplementary information Supplementary data are available at Bioinformatics online.

The BTB-Kelch protein KLHL3 is a Cullin3-dependent E3 ligase that mediates the ubiquitin-dependent degradation of kinases WNK1-4 to control blood pressure and cell volume. A crystal structure of KLHL3 has defined its binding to an acidic degron motif containing a PXXP sequence that is strictly conserved in WNK1, WNK2 and WNK4. Mutations in the second proline abrograte the interaction causing the hypertension syndrome pseudohypoaldosteronism type II. WNK3 shows a diverged degron motif containing 4 amino acid substitutions that remove the PXXP motif raising questions as to the mechanism of its binding. To understand this atypical interaction, we determined the crystal structure of the KLHL3 Kelch domain in complex with a WNK3 peptide. The electron density enabled the complete 11-mer WNK-family degron motif to be traced for the first time revealing several conserved features not captured in previous work, including additional salt bridge and hydrogen bond interactions. Overall, the WNK3 peptide adopted a conserved binding pose except for a subtle shift to accommodate bulkier amino acid substitutions at the binding interface. At the centre, the second proline was substituted by WNK3 Thr541, providing a unique phosphorylatable residue among the WNK-family degrons. Fluorescence polarisation and structural modelling experiments revealed that its phosphorylation would abrogate the KLHL3 interaction similarly to hypertension-causing mutations. Together, these data reveal how the KLHL3 Kelch domain can accommodate the binding of multiple WNK isoforms and highlight a potential regulatory mechanism for the recruitment of WNK3.

The capsid protein (Cap) is the sole structural protein and the main antigen of porcine circovirus type 2 (PCV2). Structural loops of the Cap play crucial roles in viral genome packaging, capsid assembly, and virus-host interactions. Although the molecular mechanisms are yet unknown, the carboxyl terminus (CT) of the PCV2 Cap is known to play critical roles in the evolution, pathogenesis, and proliferation of this virus. In this study, we investigated functions of CT. Removal of this loop leads to abrogation of the in vitro Cap self-assembly into virus-like particles (VLPs). Likewise, the mutated virus resists rescue from PK15 cell culture. A conserved PXXP motif in the CT is dispensable for VLP assembly and subsequent cell entry. However, its removal leads to the subsequent failure of virus rescued from PK15 cells. Furthermore, substituting either the PCV1 counterpart or an AXXA for the PXXP motif still supports virus rescue from cell culture but results in a dramatic decrease in viral titers compared with wild type. In particular, a strictly conserved residue (227K) in the CT is essential for VLP entry into PK15 cells, and its mutation to alanine greatly attenuates cell entry of the VLPs, supporting a mechanism for the failure to rescue a mutated PCV2 infectious DNA clone (K227A) from PK15 cell culture. These results suggest the CT of the PCV2 Cap plays critical roles in virus assembly, viral-host cell interaction(s), and virus propagation in vitroIMPORTANCE The carboxyl terminus (CT) of porcine circovirus type 2 (PCV2) capsid protein (Cap) was previously reported to be associated with immunorecognition, alterations of viral titer in swine sera, and pathogenicity. However, the molecular mechanisms underlying these effects remain unknown. In this study, roles of the critical residues and motifs of the CT are investigated with respect to virus-like particle (VLP) assembly, cell entry, and viral proliferation. The results revealed that the positively charged 227K of the CT is essential for both cell entry of PCV2 VLPs and virus proliferation. Our findings, therefore, suggest that the CT should be considered one of the key epitopes, recognized by neutralizing antibodies, for vaccine design and a target for drug development to prevent PCV2-associated diseases (PCVADs). Furthermore, it is important to respect the function of 227K for its role in cell entry if using either PCV2 VLPs for nanoscale DNA/drug cell delivery or using PCV2 VLPs to display a variety of foreign epitopes for immunization.

In Escherichia coli, DNA replication forks stall on average once per cell cycle. When this occurs, replisome components disengage from the DNA, exposing an intact, or nearly intact fork. Consequently, the fork structure must be regressed away from the initial impediment so that repair can occur. Regression is catalyzed by the powerful, monomeric DNA helicase, RecG. During this reaction, the enzyme couples unwinding of fork arms to rewinding of duplex DNA resulting in the formation of a Holliday junction. RecG works against large opposing forces enabling it to clear the fork of bound proteins. Following subsequent processing of the extruded junction, the PriA helicase mediates reloading of the replicative helicase DnaB leading to the resumption of DNA replication. The single-strand binding protein (SSB) plays a key role in mediating PriA and RecG functions at forks. It binds to each enzyme via linker/OB-fold interactions and controls helicase-fork loading sites in a substrate-dependent manner that involves helicase remodeling. Finally, it is displaced by RecG during fork regression. The intimate and dynamic SSB-helicase interactions play key roles in ensuring fork regression and DNA replication restart.

The E. coli single strand DNA binding protein (SSB) is essential to viability where it functions in two seemingly disparate roles: it binds to single stranded DNA (ssDNA) and to target proteins that comprise the SSB interactome. The link between these roles resides in a previously under-appreciated region of the protein known as the intrinsically disordered linker (IDL). We present a model wherein the IDL is responsible for mediating protein-protein interactions critical to each role. When interactions occur between SSB tetramers, cooperative binding to ssDNA results. When binding occurs between SSB and an interactome partner, storage or loading of that protein onto the DNA takes place. The properties of the IDL that facilitate these interactions include the presence of repeats, a putative polyproline type II helix and, PXXP motifs that may facilitate direct binding to the OB-fold in a manner similar to that observed for SH3 domain binding of PXXP ligands in eukaryotic systems.

Rho GTPases play a key role in various membrane trafficking processes. RhoU is an atypical small Rho GTPase related to Rac/Cdc42 which possesses unique N- and C-terminal domains that regulate its function and its subcellular localization. RhoU localized at the plasma membrane, on endosomes and in cell adhesion structures where it governs cell signalling, differentiation and migration. However, despite its endomembrane localization, RhoU function in vesicular trafficking has been unexplored. Here, we identified intersectins (ITSNs) as new binding partners for RhoU and showed that the second PxxP motif at the N-terminus of RhoU mediated interactions with SH3 domains of ITSNs. To evaluate the function of RhoU and ITSNs in vesicular trafficking, we used fluorescent transferrin as a cargo for uptake experiments. We showed that silencing of either RhoU or ITSN2, but not ITSN1 increased transferrin accumulation in early endosomes resulting from defect in fast vesicle recycling. Concomitantly, RhoU and ITSN2 colocalized to a subset of Rab4-positive vesicles suggesting that RhoU-ITSN2 interaction may occur on fast recycling endosomes to regulate the fate of vesicular cargos.

The phospholipase Cγ1 (PLCγ1) is essential for T-cell signaling and activation in hepatic cancer immune response, which has a regulatory Src homology 3 (SH3) domain that can specifically recognize and interact with the PXXP-containing decapeptide segment (185 QPPVPPQRPM194 , termed as SLP76185-194 peptide) of adaptor protein SLP76 following T-cell receptor ligation. The isolated peptide can only bind to the PLCγ1 SH3 domain with a moderate affinity due to lack of protein context support. Instead of the traditional natural residue mutagenesis that is limited by low structural diversity and shifted target specificity, we herein attempt to improve the peptide affinity by replacing the two key proline residues Pro187 and Pro190 of SLP76185-194 PXXP motif with nonnatural N-substituted amino acids, as the proline is the only endogenous N-substituted amino acid. The replacement would increase peptide flexibility but can restore peptide activity by establishing additional interactions with the domain. Structural analysis reveals that the domain pocket can be divided into a large amphipathic region and a small negatively charged region; they accommodate hydrophobic, aromatic, polar, and moderate-sized N-substituted amino acid types. A systematic replacement combination profile between the peptide residues Pro187 and Pro190 is created by structural modeling, dynamics simulation, and energetics analysis, from which six improved and two reduced N-substituted peptides as well as native SLP76185-194 peptide are identified and tested for their binding affinity to the recombinant protein of the human PLCγ1 SH3 domain using fluorescence-based assays. Two N-substituted peptides, SLP76185-194 (N-Leu187/N-Gln190) and SLP76185-194 (N-Thr187/N-Gln190), are designed to have high potency (Kd = 0.67 ± 0.18 and 1.7 ± 0.3 μM, respectively), with affinity improvement by, respectively, 8.5-fold and 3.4-fold relative to native peptide (Kd = 5.7 ± 1.2 μM).

Outbreaks of porcine circovirus (PCV) type 2 (PCV2)-associated diseases have caused substantial economic losses worldwide in the last 20 years. The PCV capsid protein (Cap) is the sole structural protein and main antigenic determinant of this virus. In this study, not only were phylogenetic trees reconstructed, but variations of surface structure of the PCV capsid were analysed in the course of evolution. Unique surface patterns of the icosahedral fivefold axes of the PCV2 capsid were identified and characterized, all of which were absent in PCV type 1 (PCV1). Icosahedral fivefold axes, decorated with Loops BC, HI and DE, were distinctly different between PCV2 and PCV1. Loops BC, determining the outermost surface around the fivefold axes of PCV capsids, had limited homology between Caps of PCV1 and PCV2. A conserved tyrosine phosphorylation motif in Loop HI that might be recognized by non-receptor tyrosine kinase(s) in vivo was present only in PCV2. Particularly, the concurrent presence of 60 pairs of the conserved tyrosine and a canonical PXXP motif on the PCV2 capsid surface could be a mechanism for PXXP motif binding to and activation of an SH3-domain-containing tyrosine kinase in host cells. Additionally, a conserved cysteine in Loop DE of the PCV2 Cap was substituted by an arginine in PCV1, indicating potentially distinct assembly mechanisms of the capsid in vitro between PCV1 and PCV2. Therefore, these unique patterns on the PCV2 capsid surface, absent in PCV1 isolates, might be related to cell entry, virus function and pathogenesis.

Hepatitis C virus (HCV) non-structural 5A (NS5A) protein is associated with a wide variety of host signaling pathways by binding to C-terminal polyproline (PxxP) motifs of various proteins. In this study, we used yeast two-hybrid analysis and a GST pull-down assay to screen a novel NS5A interacting protein and elucidate the binding site and cellular signaling by focusing on recombinant human epidermal growth factor (rhEGF)-mediated ERK1/2 activation. Screening a liver cDNA library revealed that h-prune, a member of the DHH (Asp-His-His) protein superfamily, directly interacted with HCV NS5A C-terminus. In particular, a mutation of five proline amino acids to alanine in this region revealed that these two proteins produced strong interaction through this domain. It is known that h-prune possesses a highly conserved DHH motif, which has exopolyphosphatase activity that accelerates hydrolysis of inorganic polyphosphate. A time-chasing analysis after rhEGF treatment demonstrated that h-prune overexpression almost restored NS5A-mediated attenuation of ERK1/2 phosphorylation, but h-prune itself did not alter this signaling. Although the detailed mechanisms need to be clarified, this study demonstrates that h-prune interacts directly with the PxxP motif of the HCV NS5A C-terminus and that this binding alters the rhEGF-mediated ERK1/2 signaling cascade in liver cells.

Dynamin is a GTPase that mediates vesicle fission during synaptic vesicle endocytosis. Its long C-terminal proline-rich domain contains 13 PXXP motifs, which orchestrate its interactions with multiple proteins. The SH3 domains of syndapin and endophilin bind the PXXP motifs called Site 2 and 3 (Pro-786-Pro-793) at the N-terminal end of the proline-rich domain, whereas the amphiphysin SH3 binds Site 9 (Pro-833-Pro-836) toward the C-terminal end. In some proteins, SH3/peptide interactions also involve short distance elements, which are 5-15 amino acid extensions flanking the central PXXP motif for high affinity binding. Here we found two previously unrecognized elements in the central and the C-terminal end of the dynamin proline-rich domain that account for a significant increase in syndapin binding affinity compared with a previously reported Site 2 and Site 3 PXXP peptide alone. The first new element (Gly-807-Gly-811) is short distance element on the C-terminal side of Site 2 PXXP, which might contact a groove identified under the RT loop of the SH3 domain. The second element (Arg-838-Pro-844) is located about 50 amino acids downstream of Site 2. These two elements provide additional specificity to the syndapin SH3 domain outside of the well described polyproline-binding groove. Thus, the dynamin/syndapin interaction is mediated via a network of multiple contacts outside the core PXXP motif over a previously unrecognized extended region of the proline-rich domain. To our knowledge this is the first example among known SH3 interactions to involve spatially separated and extended long-range elements that combine to provide a higher affinity interaction.

Mammalian Fat1 is a giant atypical cadherin/tumor suppressor involved in the regulation of cellular orientation, migration, and growth. Fat1 is implicated in the development of the brain, eye, and kidney. Altered expression or mutations of FAT1 are also associated with cancer and facioscapulohumeral muscular dystrophy (FSHD). Yet, the mechanistic functions of this pathway remain incompletely understood. Here, we report the identification of Sorbin-homology (SoHo) proteins as novel interaction partners of Fat1 by virtue of a yeast-two-hybrid screen. SoHo proteins play diverse roles as adaptor proteins in cell signaling, cell adhesion and sarcomere architecture, including altered expression in cancer and FSHD. Specifically, we found SoHo proteins CAP/ponsin-1 and -2 (Sorbs1) and ArgBP2 (Sorbs2) to interact with the cytoplasmic domain of Fat1. We mapped the interaction to a prolin-rich classic type II PXXP motif within Fat1 and to the three Src-homology (SH3) domains within SoHo proteins using mutant expression in yeast, pulldown assays, and cell culture. Functionally, endogenous ponsin-2 expression of NRK-52E cells at cellular leading edges was lost upon knockdown of Fat1. In summary, our data point to an interaction of Fat1 with SoHo proteins that is able to recruit SoHo proteins to sites of Fat1 expression.

Porcine circovirus type 2 (PCV2) causes huge economic losses in the global swine industry and has a complex and poorly understood virus-host interaction mechanism. We reported that the C-terminal of the capsid protein of all PCV2 isolates shared a strictly conserved PXXP motif that may interact with SH3 domain-containing tyrosine kinases; however, its roles in PCV2 cell entry and replication remain unknown. In this study, we determined that mRNA levels of two SH3 domain-containing tyrosine kinases family (Abl and Src) had distinct profiles (wild-type and PXXP-mutated) during PCV2 infections of PK15 cells. Therefore, we hypothesized that activities of tyrosine kinases (Abl and Fyn) in PK15 cells may be hijacked by PCV2 via its PXXP motif of the Cap, to favor virus replication. Specific inhibitors PP2 of Lck/Fyn and STI-571 of Abl family kinases decreased viral production through suppression of DNA and Cap synthesis at the replication stage. However, based on indirect immunofluorescence assay (IFA), entry of PCV2 virus-like particles (VLPs) into PK15 cells was not altered. Elucidating mechanisms of PCV2-host interactions should provide new insights for development of new compounds to prevent or reduce PCV2 infections.

The ORF3 protein of hepatitis E virus (HEV) contains a “PSAP” amino acid late domain motif, which allows for interaction with the endosomal sorting complexes required for transport (ESCRT) pathway aiding virion release. Late domain motifs are interchangeable with other viral late domain motifs in several enveloped viruses, however, it remains unknown whether HEV shares this functional interchangeability and what implications this might have on viral replication. In this study, by substituting heterologous late domain motifs (PPPY, YPDL, and PSAA) for the HEV ORF3 late domain (PSAP), we demonstrated that deviation from the PSAP motif reduces virus release as measured by viral RNA in culture media. Virus release could not be restored by insertion of a heterologous late domain motif or by supplying wild-type ORF3 in trans, suggesting that the HEV PSAP motif is required for viral exit which cannot be bypassed by the use of alternative heterologous late domains.

The Slit-Robo GTPase-activating protein 3 (srGAP3) has been implicated in different critical aspects of neuronal development. These findings have mainly been based on the characterisation of the three conserved globular N-terminal domains, while the function of the C-terminal region (CTR) is still unknown. We show that this predicted unstructured region acts as an adaptor by binding to the endocytic proteins Amphiphysin, Endophilin-A2, Endophilin-A1, as well as the Ras signalling protein Grb2. All these interactions depend on a single proline-rich motif in the CTR and the Src-homology 3 domains of the binding partners. Via these interactions srGAP3 could link receptor signalling events to the endocytic machinery.

Abelson tyrosine kinase (Abl) is a key regulator of actin-related morphogenetic processes including axon guidance, where it functions downstream of several guidance receptors. While the long C-terminal domain (CTD) of Abl is required for function, its role is poorly understood. Here, a battery of mutants of Drosophila Abl was created that systematically deleted large segments of the CTD from Abl or added them back to the N-terminus alone. The functionality of these Abl transgenes was assessed through rescue of axon guidance defects and adult lethality in Abl loss-of-function, as well as through gain-of-function effects in sensitized slit or frazzled backgrounds that perturb midline guidance in the Drosophila embryonic nerve cord. Two regions of the CTD play important and distinct roles, but additive effects for other regions were also detected. The first quarter of the CTD, including a conserved PxxP motif and its surrounding sequence, regulates Abl function while the third quarter localizes Abl to axons. These regions feature long stretches of intrinsically disordered sequence typically found in hub proteins and are associated with diverse protein-protein interactions. Thus, the CTD of Abl appears to use these disordered regions to establish a variety of different signaling complexes required during formation of axon tracts.

Spon2 is a proto-oncogene matrix protein that plays an essential role in the tumorigenesis and metastasis of gastric cancer. The protein has recently been found to function as a guanine nucleotide exchange factor through the activation of RhoGTPase. Here, computational modeling and bioinformatics analysis were employed to investigate the molecular mechanism and biological implication underlying Spon2 autoinhibition. It is revealed that the binding of PxxP motif to SH domain can stabilize the intramolecular interaction between the N-terminal helix and DH domain of Spon2, thus shifting the protein into an autoinhibitory state. Here, we proposed releasing Spon2 autoinhibition by targeting SH domain with competitive peptide ligands. To verify this notion, the PxxP sequence was adopted as the start to derive an array of efficient SH binders by using a structure-based rational design strategy, which were then substantiated with fluorescence spectroscopy analysis and guanine nucleotide exchange test. Consequently, the obtained peptide ligands were determined to have a moderate or high affinity for SH domain; they can also enhance Spon2 exchange activity by 1.2–6.1 folds, exhibiting a significant correlation with their SH-binding affinity (Pearson’s coefficient = 0.92). In addition, neutral substitution of conserved residues in a high-affinity peptide ligand can largely reduce its Spon2-activating potency, confirming that the designed peptide activates Spon2 by competitively disrupting SH–PxxP interaction.

Bovine leukemia virus (BLV) was detected and genotyped in a population of 201 dairy cattle from central Mexico. Using a commercial indirect enzyme-linked immunosorbent assay (iELISA) kit, 118 polymerase chain reaction (PCR)-positive and BLV antibody-positive samples were identified; the concordance between tests was substantial. A phylogenetic study of 27 partial sequences of the env gene gp30 was performed. Four mutations were detected involving the PXXP motif in the cytoplasmic domain of the transmembrane protein. This study provided evidence of the efficacy of PCR for the detection of BLV and demonstrated the presence of genotype 1 BLV in Mexico.

Indolicidin (IR13), a 13-residue antimicrobial peptide from the cathelicidin family, is known to exhibit a broad spectrum of antimicrobial activity against various microorganisms. This peptide inhibits bacterial DNA synthesis resulting in cell filamentation. However, the precise mechanism remains unclear and requires further investigation. The central PWWP motif of IR13 provides a unique structural element that can wrap around, and thus stabilize, duplex B-type DNA structures. Replacements of the central Trp-Trp pair with Ala-Ala, His-His, or Phe-Phe residues in the PxxP motif significantly affects the ability of the peptide to stabilize duplex DNA. Results of microscopy studies in conjunction with spectroscopic data confirm that the DNA duplex is stabilized by IR13, thereby inhibiting DNA replication and transcription. In this study we provide high-resolution structural information on the interaction between indolicidin and DNA, which will be beneficial for the design of novel therapeutic antibiotics based on peptide scaffolds.