Triscysteine disulfide-directing motifs enabling design and discovery of multicyclic peptide binders.

Peptides are valuable for therapeutic development, with multicyclic peptides showing promise in mimicking antigen-binding potency of antibodies. However, our capability to engineer multicyclic peptide scaffolds, particularly for the construction of large combinatorial libraries, is still limited. Here, we study the interplay of disulfide pairing between three biscysteine motifs, and designed a range of triscysteine motifs with unique disulfide-directing capability for regulating the oxidative folding of multicyclic peptides. We demonstrate that incorporating these motifs into random sequences allows the design of disulfide-directed multicyclic peptide (DDMP) libraries with up to four disulfide bonds, which have been applied for the successful discovery of peptide binders with nanomolar affinity to several challenging targets. This study encourages the use of more diverse disulfide-directing motifs for creating multicyclic peptide libraries and opens an avenue for discovering functional peptides in sequence and structural space beyond existing peptide scaffolds, potentially advancing the field of peptide drug discovery.

Substrate specificity of a branch of aromatic dioxygenases determined by three distinct motifs.

The inversion of substrate size specificity is an evolutionary roadblock for proteins. The Duf4243 dioxygenases GedK and BTG13 are known to catalyze the aromatic cleavage of bulky tricyclic hydroquinone. In this study, we discover a Duf4243 dioxygenase PaD that favors small monocyclic hydroquinones from the penicillic-acid biosynthetic pathway. Sequence alignments between PaD and GedK and BTG13 suggest PaD has three additional motifs, namely motifs 1-3, distributed at different positions in the protein sequence. X-ray crystal structures of PaD with the substrate at high resolution show motifs 1-3 determine three loops (loops 1-3). Most intriguing, loops 1-3 stack together at the top of the pocket, creating a lid-like tertiary structure with a narrow channel and a clearly constricted opening. This drastically changes the substrate specificity by determining the entry and binding of much smaller substrates. Further genome mining suggests Duf4243 dioxygenases with motifs 1-3 belong to an evolutionary branch that is extensively involved in the biosynthesis of natural products and has the ability to degrade diverse monocyclic hydroquinone pollutants. This study showcases how natural enzymes alter the substrate specificity fundamentally by incorporating new small motifs, with a fixed overall scaffold-architecture. It will also offer a theoretical foundation for the engineering of substrate specificity in enzymes and act as a guide for the identification of aromatic dioxygenases with distinct substrate specificities.

Molecular mechanism of IKK catalytic dimer docking to NF-κB substrates.

The inhibitor of κB (IκB) kinase (IKK) is a central regulator of NF-κB signaling. All IKK complexes contain hetero- or homodimers of the catalytic IKKß and/or IKKα subunits. Here, we identify a YDDΦxΦ motif, which is conserved in substrates of canonical (IκBα, IκBß) and alternative (p100) NF-κB pathways, and which mediates docking to catalytic IKK dimers. We demonstrate a quantitative correlation between docking affinity and IKK activity related to IκBα phosphorylation/degradation. Furthermore, we show that phosphorylation of the motif's conserved tyrosine, an event previously reported to promote IκBα accumulation and inhibition of NF-κB gene expression, suppresses the docking interaction. Results from integrated structural analyzes indicate that the motif binds to a groove at the IKK dimer interface. Consistently, suppression of IKK dimerization also abolishes IκBα substrate binding. Finally, we show that an optimized bivalent motif peptide inhibits NF-κB signaling. This work unveils a function for IKKα/ß dimerization in substrate motif recognition.

The C-terminal amino acid motifs of NS1 protein affect the replication and virulence of naturally NS-truncated H1N1 canine influenza virus.

The vast majority of data obtained from sequence analysis of influenza A viruses (IAVs) have revealed that nonstructural 1 (NS1) proteins from H1N1 swine, H3N8 equine, H3N2 avian and the correspondent subtypes from dogs have a conserved four C-terminal amino acid motif when independent cross-species transmission occurs between these species. To test the influence of the C-terminal amino acid motifs of NS1 protein on the replication and virulence of IAVs, we systematically generated 7 recombinants, which carried naturally truncated NS1 proteins, and their last four C-terminal residues were replaced with PEQK and SEQK (for H1N1), EPEV and KPEI (for H3N8) and ESEV and ESEI (for H3N2) IAVs. Another recombinant was generated by removing the C-terminal residues by reverse genetics. Remarkably, the ESEI and KPEI motifs circulating in canines largely contributed efficient replication in cultured cells and these had enhanced virulence. In contrast, the avian ESEV motif was only responsible for high pathogenicity in mice. We examined the effects of these motifs upon interferon (IFN) induction. The 7 mutant viruses replicated in vitro in an IFN-independent manner, and the canine SEQK motif was able to induced higher levels of IFN-ß in human cell lines. These findings shed further new light on the role of the four C-terminal residues in replication and virulence of IAVs and suggest that these motifs can modulate viral replication in a species-specific manner.

Conserved regulatory motifs in the juxtamembrane domain and kinase N-lobe revealed through deep mutational scanning of the MET receptor tyrosine kinase domain.

MET is a receptor tyrosine kinase (RTK) responsible for initiating signaling pathways involved in development and wound repair. MET activation relies on ligand binding to the extracellular receptor, which prompts dimerization, intracellular phosphorylation, and recruitment of associated signaling proteins. Mutations, which are predominantly observed clinically in the intracellular juxtamembrane and kinase domains, can disrupt typical MET regulatory mechanisms. Understanding how juxtamembrane variants, such as exon 14 skipping (METΔEx14), and rare kinase domain mutations can increase signaling, often leading to cancer, remains a challenge. Here, we perform a parallel deep mutational scan (DMS) of the MET intracellular kinase domain in two fusion protein backgrounds: wild-type and METΔEx14. Our comparative approach has revealed a critical hydrophobic interaction between a juxtamembrane segment and the kinase ⍺C-helix, pointing to potential differences in regulatory mechanisms between MET and other RTKs. Additionally, we have uncovered a ß5 motif that acts as a structural pivot for the kinase domain in MET and other TAM family of kinases. We also describe a number of previously unknown activating mutations, aiding the effort to annotate driver, passenger, and drug resistance mutations in the MET kinase domain.

Replication licensing regulated by a short linear motif within an intrinsically disordered region of origin recognition complex.

In eukaryotes, the origin recognition complex (ORC) faciliates the assembly of pre-replicative complex (pre-RC) at origin DNA for replication licensing. Here we show that the N-terminal intrinsically disordered region (IDR) of the yeast Orc2 subunit is crucial for this process. Removing a segment (residues 176-200) from Orc2-IDR or mutating a key isoleucine (194) significantly inhibits replication initiation across the genome. These Orc2-IDR mutants are capable of assembling the ORC-Cdc6-Cdt1-Mcm2-7 intermediate, which exhibits impaired ATP hydrolysis and fails to be convered into the subsequent Mcm2-7-ORC complex and pre-RC. These defects can be partially rescued by the Orc2-IDR peptide. Moreover, the phosphorylation of this Orc2-IDR region by S cyclin-dependent kinase blocks its binding to Mcm2-7 complex, causing a defective pre-RC assembly. Our findings provide important insights into the multifaceted roles of ORC in supporting origin licensing during the G1 phase and its regulation to restrict origin firing within the S phase.

The Archetypal Gamma-Core Motif of Antimicrobial Cys-Rich Peptides Inhibits H+-ATPases in Target Pathogens.

Human lactoferrin (hLf) is an innate host defense protein that inhibits microbial H+-ATPases. This protein includes an ancestral structural motif (i.e., γ-core motif) intimately associated with the antimicrobial activity of many natural Cys-rich peptides. Peptides containing a complete γ-core motif from hLf or other phylogenetically diverse antimicrobial peptides (i.e., afnA, SolyC, PA1b, PvD1, thanatin) showed microbicidal activity with similar features to those previously reported for hLf and defensins. Common mechanistic characteristics included (1) cell death independent of plasma membrane (PM) lysis, (2) loss of intracellular K+ (mediated by Tok1p K+ channels in yeast), (3) inhibition of microbicidal activity by high extracellular K+, (4) influence of cellular respiration on microbicidal activity, (5) involvement of mitochondrial ATP synthase in yeast cell death processes, and (6) increment of intracellular ATP. Similar features were also observed with the BM2 peptide, a fungal PM H+-ATPase inhibitor. Collectively, these findings suggest host defense peptides containing a homologous γ-core motif inhibit PM H+-ATPases. Based on this discovery, we propose that the γ-core motif is an archetypal effector involved in the inhibition of PM H+-ATPases across kingdoms of life and contributes to the in vitro microbicidal activity of Cys-rich antimicrobial peptides.

Design Glutamate Dehydrogenase for Nonaqueous System by Motifs Reassembly and Interaction Network Analysis.

Glutamate dehydrogenases (GDH) serve as the key regulated enzyme that links protein and carbohydrate metabolism. Combined with motif reassembly and mutation, novel GDHs were designed. Motif reassembly of thermophilic GDH and malate dehydrogenase aims to overcome stability and activity tradeoff in nonaqueous systems. Structural compatibility and dynamic cooperation of the designed AaDHs were studied by molecular dynamics simulation. Furthermore, multipoint mutations improved its catalytic activity for unnatural substrates. Amino acid interaction network analysis indicated that the high density of hydrogen-bonded salt bridges is beneficial to the stability. Finally, the experimental verification determines the kinetics of AaDHs in a nonaqueous system. The activity of Aa05 was increased by 1.78-fold with ionic liquid [EMIM]BF4. This study presents the strategy of a combination of rigid motif assembly and mutations of active sites for robust dehydrogenases with high activity in the nonaqueous system, which overcomes the activity-stability tradeoff effect.

The Chlamydia effector IncE employs two short linear motifs to reprogram host vesicle trafficking.

Chlamydia trachomatis, a leading cause of bacterial sexually transmitted infections, creates a specialized intracellular replicative niche by translocation and insertion of a diverse array of effectors (Incs [inclusion membrane proteins]) into the inclusion membrane. Here, we characterize IncE, a multifunctional Inc that encodes two non-overlapping short linear motifs (SLiMs) within its short cytosolic C terminus. The proximal SLiM, by mimicking just a small portion of an R-N-ethylmaleimide-sensitive factor adaptor protein receptor (SNARE) motif, binds and recruits syntaxin (STX)7- and STX12-containing vesicles to the inclusion. The distal SLiM mimics the sorting nexin (SNX)5 and SNX6 cargo binding site to recruit SNX6-containing vesicles to the inclusion. By simultaneously binding two distinct vesicle classes, IncE brings these vesicles in close apposition with each other at the inclusion to facilitate C. trachomatis intracellular development. Our work suggests that Incs may have evolved SLiMs to enable rapid evolution in a limited protein space to disrupt host cell processes.

mTORC1 phosphorylates and stabilizes LST2 to negatively regulate EGFR.

TORC1 (target of rapamycin complex 1) is a highly conserved protein kinase that plays a central role in regulating cell growth. Given the role of mammalian TORC1 (mTORC1) in metabolism and disease, understanding mTORC1 downstream signaling and feedback loops is important. mTORC1 recognizes some of its substrates via a five amino acid binding sequence called the TOR signaling (TOS) motif. mTORC1 binding to a TOS motif facilitates phosphorylation of a distinct, distal site. Here, we show that LST2, also known as ZFYVE28, contains a TOS motif (amino acids 401 to 405) and is directly phosphorylated by mTORC1 at serine 670 (S670). mTORC1-mediated S670 phosphorylation promotes LST2 monoubiquitination on lysine 87 (K87). Monoubiquitinated LST2 is stable and displays a broad reticular distribution. When mTORC1 is inactive, unphosphorylated LST2 is degraded by the proteasome. The absence of LST2 enhances EGFR (epidermal growth factor receptor) signaling. We propose that mTORC1 negatively feeds back on its upstream receptor EGFR via LST2.

CagA 3' region polymorphism of Helicobacter pylori and its association with chronic gastritis in the Chinese population.

Introduction. Cytotoxin-associated gene A (CagA) from Helicobacter pylori is highly related to chronic gastritis. Tyrosine phosphorylation of Glu-Pro-Ile-Tyr-Ala (EPIYA) motifs from CagA determines the pathogenicity of H. pylori.Gap statement. The precise amino acid variations surrounding the EPIYA motifs and their correlation with clinical outcomes have been poorly explored.Aim. The purpose of this study was to examine the CagA 3' region polymorphism of H. pylori and its association with chronic gastritis in the Chinese population.Method. A total of 86 cagA-positive H. pylori strains were isolated from patients with chronic gastritis in two different hospitals in Beijing, PR China. Genomic DNA was extracted commercial kits, and the cagA 3' variable region of H. pylori was amplified by polymerase chain reaction (PCR). The PCR products were sequenced and analysed using the CLC Sequence Viewer, BioEdit, and WebLogo 3.Results. Two hundred and fifty-nine EPIYA motifs were identified from cagA-positive H. pylori strains. Notably, EPIYA-B exhibited a higher frequency of variation in comparison to EPIYA-A, EPIYA-C, and EPIYA-D. The prevalent sequences for East-Asian-type CagA were QVNK and TIDF, while KVNK and TIDD were most commonly observed for Western-type CagA. The CRPIA motifs of East-Asian-type CagA and Western-type CagA varied at positions 4, 6, 7, 8, and 10. CagA-ABD (73.2%) was the most prevalent type, followed by CagA-ABC (18.6%) and CagA-AB (3.4%). The ratio of CagA-ABD was observed to be higher in cases of chronic non-atrophic gastritis with erosive (NAGE) or chronic atrophic gastritis (AG) compared to chronic non-atrophic gastritis (NAG), and the difference was found to be statistically significant (χ2=59.000/64.000, P<0.001).Conclusions. The EPIYA segments of Western-type CagA and East-Asian-type CagA differ significantly and the presence of CagA-ABD may be associated with severe chronic gastritis from this study.

In Silico Assessment of LIR Motif-Containing Proteins Binding to ATG8-Family Proteins.

Selective autophagic degradation of cellular components has been shown to be mediated by the interaction of LIR motif-containing proteins with ATG8-family proteins. Here, we present a detailed methodology for the in silico evaluation of potential binding between LIR motif-containing proteins and ATG8-family proteins. We visualize AlphaFold-predicted protein complexes using PyMOL to assess potential interactions, providing an effective computational tool for this purpose.

Determination of hATG8 Binding Selectivity of AIM (Autophagy-Interacting Motif) Peptides Using Native Electrospray Ionization Mass Spectrometry.

Establishing the hATG8 binding selectivity of AIM (autophagy-interacting motif) sequences found within autophagy system proteins provides insights into their biological roles, and in the case of disease-associated AIM mutations, potential pathophysiological mechanisms. Given the sometimes small differences in affinity for an individual AIM amongst the six hATG8 proteins, establishing AIM preferences can be experimentally challenging. We describe a native mass spectrometry method that is suitable for detecting such differences, using synthetic AIM peptides and recombinant hATG8 proteins, to probe hATG8-AIM interactions in the gas phase. Binding preferences of a single AIM peptide against multiple hATG8s, or two AIM peptides against a single hATG8 (e.g., wild-type versus mutant AIM), may be determined.

The ß-d-manno-heptoses are immune agonists across kingdoms.

Bacterial small molecule metabolites such as adenosine-diphosphate-d-glycero-ß-d-manno-heptose (ADP-heptose) and their derivatives act as effective innate immune agonists in mammals. We show that functional nucleotide-diphosphate-heptose biosynthetic enzymes (HBEs) are distributed widely in bacteria, archaea, eukaryotes, and viruses. We identified a conserved STTR5 motif as a hallmark of heptose nucleotidyltransferases that can synthesize not only ADP-heptose but also cytidine-diphosphate (CDP)- and uridine-diphosphate (UDP)-heptose. Both CDP- and UDP-heptoses are agonists that trigger stronger alpha-protein kinase 1 (ALPK1)-dependent immune responses than ADP-heptose in human and mouse cells and mice. We also produced ADP-heptose in archaea and verified its innate immune agonist functions. Hence, the ß-d-manno-heptoses are cross-kingdom, small-molecule, pathogen-associated molecular patterns that activate the ALPK1-dependent innate immune signaling cascade.

The Ribosome Assembly Factor LSG1 Interacts with Vesicle-Associated Membrane Protein-Associated Proteins (VAPs).

LSG1 is a conserved GTPase involved in ribosome assembly. It is required for the eviction of the nuclear export adapter NMD3 from the pre-60S subunit in the cytoplasm. In human cells, LSG1 has also been shown to interact with vesicle-associated membrane protein-associated proteins (VAPs) that are found primarily on the endoplasmic reticulum. VAPs interact with a large host of proteins which contain FFAT motifs (two phenylalanines (FF) in an acidic tract) and are involved in many cellular functions including membrane traffic and regulation of lipid transport. Here, we show that human LSG1 binds to VAPs via a noncanonical FFAT-like motif. Deletion of this motif specifically disrupts the localization of LSG1 to the ER, without perturbing LSG1-dependent recycling of NMD3 in cells or modulation of LSG1 GTPase activity in vitro.

Extracellular domain, hinge, and transmembrane determinants affecting surface CD4 expression of a novel anti-HIV chimeric antigen receptor (CAR) construct.

Chimeric antigen receptor (CAR)-T cells have demonstrated clinical potential, but current receptors still need improvements to be successful against chronic HIV infection. In this study, we address some requirements of CAR motifs for strong surface expression of a novel anti-HIV CAR by evaluating important elements in the extracellular, hinge, and transmembrane (TM) domains. When combining a truncated CD4 extracellular domain and CD8α hinge/TM, the novel CAR did not express extracellularly but was detectable intracellularly. By shortening the CD8α hinge, CD4-CAR surface expression was partially recovered and addition of the LYC motif at the end of the CD8α TM fully recovered both intracellular and extracellular CAR expression. Mutation of LYC to TTA or TTC showed severe abrogation of CAR expression by flow cytometry and confocal microscopy. Additionally, we determined that CD4-CAR surface expression could be maximized by the removal of FQKAS motif at the junction of the extracellular domain and the hinge region. CD4-CAR surface expression also resulted in cytotoxic CAR T cell killing of HIV Env+ target cells. In this study, we identified elements that are crucial for optimal CAR surface expression, highlighting the need for structural analysis studies to establish fundamental guidelines of CAR designs.

Molecular mechanism and functional significance of Wapl interaction with the Cohesin complex.

The ring-shaped Cohesin complex, consisting of core subunits Smc1, Smc3, Scc1, and SA2 (or its paralog SA1), topologically entraps two duplicated sister DNA molecules to establish sister chromatid cohesion in S-phase. It remains largely elusive how the Cohesin release factor Wapl binds the Cohesin complex, thereby inducing Cohesin disassociation from mitotic chromosomes to allow proper resolution and separation of sister chromatids. Here, we show that Wapl uses two structural modules containing the FGF motif and the YNARHWN motif, respectively, to simultaneously bind distinct pockets in the extensive composite interface between Scc1 and SA2. Strikingly, only when both docking modules are mutated, Wapl completely loses the ability to bind the Scc1-SA2 interface and release Cohesin, leading to erroneous chromosome segregation in mitosis. Surprisingly, Sororin, which contains a conserved FGF motif and functions as a master antagonist of Wapl in S-phase and G2-phase, does not bind the Scc1-SA2 interface. Moreover, Sgo1, the major protector of Cohesin at mitotic centromeres, can only compete with the FGF motif but not the YNARHWN motif of Wapl for binding Scc1-SA2 interface. Our data uncover the molecular mechanism by which Wapl binds Cohesin to ensure precise chromosome segregation.

Probing the effects of single point mutations in the GKWWRPS motif on the PNAIG motif within Loop 2 of sclerostin (SOST) using in-silico techniques.

Sclerostin (SOST), a Wnt signaling pathway inhibitor, is involved in the pathogenesis of skeletal disorders. This study investigated the impact of the GKWWRPS motif on the PNAIG motif in Loop 2 of SOST, which is accountable for the interactions with the LRP6 protein that triggers the down-regulation of the Wnt signaling pathway. Single amino acid mutations on the GKWWRPS motif, hypothesized to have a probable stabilization effect towards the PNAIG motif, led to a significant reduction in the primary interactions between the SOST and LRP6 proteins. Protein-protein docking and molecular dynamic studies were conducted to investigate the role of the motif. The study found that a solitary mutation in the GKWWRPS motif significantly reduced the primary interactions between SOST and LRP6 proteins, except for probable cold-spot residues. The study's findings establish the GKWWRPS motif as a promising target for therapeutic interventions. Based on the obtained results, it can be inferred that alterations implemented within the GKWWRPS motif could lead to the destabilization of the PNAIG motif, which would directly modulate the interactions between the SOST and LRP6 proteins. The present investigation thus presents novel opportunities in the field of anti-sclerostin interventions.

Interaction between SARS-CoV PBM and Cellular PDZ Domains Leading to Virus Virulence.

The interaction between SARS-CoV PDZ-binding motifs (PBMs) and cellular PDZs is responsible for virus virulence. The PBM sequence present in the 3a and envelope (E) proteins of SARS-CoV can potentially bind to over 400 cellular proteins containing PDZ domains. The role of SARS-CoV 3a and E proteins was studied. SARS-CoVs, in which 3a-PBM and E-PMB have been deleted (3a-PBM-/E-PBM-), reduced their titer around one logarithmic unit but still were viable. In addition, the absence of the E-PBM and the replacement of 3a-PBM with that of E did not allow the rescue of SARS-CoV. E protein PBM was necessary for virulence, activating p38-MAPK through the interaction with Syntenin-1 PDZ domain. However, the presence or absence of the homologous motif in the 3a protein, which does not bind to Syntenin-1, did not affect virus pathogenicity. Mutagenesis analysis and in silico modeling were performed to study the extension of the PBM of the SARS-CoV E protein. Alanine and glycine scanning was performed revealing a pair of amino acids necessary for optimum virus replication. The binding of E protein with the PDZ2 domain of the Syntenin-1 homodimer induced conformational changes in both PDZ domains 1 and 2 of the dimer.