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Advances in sequencing technologies have led to a rapid growth of public protein sequence databases, whereby the fraction of proteins with experimentally verified function continuously decreases. This problem is currently addressed by automated functional annotations with computational tools, which however lack the accuracy of experimental approaches and are susceptible to error propagation. Here, we present an approach that combines the efficiency of functional annotation by in silico methods with the rigor of enzyme characterization in vitro. First, a thorough experimental analysis of a representative enzyme of a group of homologues is performed which includes a focused alanine scan of the active site to determine a fingerprint of function‐determining residues. In a second step, this fingerprint is used in combination with a sequence similarity network to identify putative isofunctional enzymes among the homologues. Using this approach in a proof‐of‐principle study, homologues of the histidinol phosphate phosphatase (HolPase) from Pseudomonas aeruginosa, many of which were annotated as phosphoserine phosphatases, were predicted to be HolPases. This functional annotation of the homologues was verified by in vitro testing of several representatives and an analysis of the occurrence of annotated HolPases in the corresponding phylogenetic groups. Moreover, the application of the same approach to the homologues of the HolPase from the archaeon Nitrosopumilus maritimus, which is not related to the HolPase from P. aeruginosa and was newly discovered in the course of this work, led to the annotation of the putative HolPase from various archaeal species. [ABSTRACT FROM AUTHOR]

Serine hydroxymethyltransferase 2 (SHMT2) has garnered significant attention as a critical catalytic regulator of the serine/glycine pathway in the one-carbon metabolism of cancer cells. Despite its potential as an anti-cancer target, only a limited number of inhibitors have been identified so far. In this study, we employed seven different scoring functions and skeleton clustering to screen the ChemDiv database for 38 compounds, 20 of which originate from the same skeleton structure. The most significant residues from SHMT2 and chemical groups from the inhibitors were identified using ASGBIE (Alanine Scanning with Generalized Born model and Interaction Entropy), and the binding energy of each residue was quantitatively determined, revealing the essential features of the protein–inhibitor interaction. The two most important contributing residues are TYR105 and TYR106 of the B chain followed by LEU166 and ARG425 of the A chain. The findings will be greatly helpful in developing a thorough comprehension of the binding mechanisms involved in drug–SHMT2 interactions and offer valuable direction for designing more potent inhibitors.

Serine hydroxymethyltransferase 2 (SHMT2) has garnered significant attention as a critical catalytic regulator of the serine/glycine pathway in the one-carbon metabolism of cancer cells. Despite its potential as an anti-cancer target, only a limited number of inhibitors have been identified so far. In this study, we employed seven different scoring functions and skeleton clustering to screen the ChemDiv database for 38 compounds, 20 of which originate from the same skeleton structure. The most significant residues from SHMT2 and chemical groups from the inhibitors were identified using ASGBIE (Alanine Scanning with Generalized Born model and Interaction Entropy), and the binding energy of each residue was quantitatively determined, revealing the essential features of the protein–inhibitor interaction. The two most important contributing residues are TYR105 and TYR106 of the B chain followed by LEU166 and ARG425 of the A chain. The findings will be greatly helpful in developing a thorough comprehension of the binding mechanisms involved in drug–SHMT2 interactions and offer valuable direction for designing more potent inhibitors. [ABSTRACT FROM AUTHOR]

ABSTRACTMonoclonal antibodies have become an important class of therapeutics in the last 30 years. Because the mechanism of action of therapeutic antibodies is intimately linked to their binding epitopes, identification of the epitope of an antibody to the antigen plays a central role during antibody drug development. The gold standard of epitope mapping, X-ray crystallography, requires a high degree of proficiency with no guarantee of success. Here, we evaluated six widely used alternative methods for epitope identification (peptide array, alanine scan, domain exchange, hydrogen-deuterium exchange, chemical cross-linking, and hydroxyl radical footprinting) in five antibody-antigen combinations (pembrolizumab+PD1, nivolumab+PD1, ipilimumab+CTLA4, tremelimumab+CTLA4, and MK-5890+CD27). The advantages and disadvantages of each technique are demonstrated by our data and practical advice on when and how to apply specific epitope mapping techniques during the drug development process is provided. Our results suggest chemical cross-linking most accurately identifies the epitope as defined by crystallography.

The d-amino acid oxidase (DAAO) is pivotal in obtaining optically pure l-glufosinate (l-PPT) by converting d-glufosinate (d-PPT) to its deamination product. We screened and designed a Rasamsonia emersonii DAAO (ReDAAO), making it more suitable for oxidizing d-PPT. Using Caver 3.0, we delineated three substrate binding pockets and, via alanine scanning, identified nearby key residues. Pinpointing key residues influencing activity, we applied virtual saturation mutagenesis (VSM), and experimentally validated mutants which reduced substrate binding energy. Analysis of positive mutants revealed elongated side-chain prevalence in substrate binding pocket periphery. Although computer-aided approaches can rapidly identify advantageous mutants and guide further design, the mutations obtained in the first round may not be suitable for combination with other advantageous mutations. Therefore, each round of combination requires reasonable iteration. Employing VSM-assisted screening multiple times and after four rounds of combining mutations, we ultimately obtained a mutant, N53V/F57Q/V94R/V242R, resulting in a mutant with a 5097% increase in enzyme activity compared to the wild type. It provides valuable insights into the structural determinants of enzyme activity and introduces a novel rational design procedure., (© 2024 Wiley‐VCH GmbH.)

Proinflammatory chemokine ligand 26 (CCL26, eotaxin-3) mediates transendothelial cell migration of eosinophils by binding and activating the G-protein-coupled (GPC) chemokine receptor 3 on the surface of eosinophilic cells. Here we have investigated the role of glycosaminoglycans (GAGs) as potential co-receptors in the process of CCL26-induced eosinophil chemotaxis. For this purpose, we have first identified the GAG-binding site of CCL26 by a site-directed mutagenesis approach in the form of an alanine screening. A panel of GAG-binding-deficient mutants has been designed, generated, and analyzed with respect to their binding affinities to heparan sulphate (HS) by isothermal fluorescence titration studies. This showed that basic amino acids in the α-helical part of CCL26 are strongly involved in GAG-binding. In chemotaxis experiments, we found that decreased GAG-binding affinity correlated with decreased chemotactic activity, which indicates an involvement of GAGs in eosinophil migration. This was further proven by the negative impact of heparinase III treatment and, independently, by the incubation of eosinophils with an anti heparan sulfate antibody. We finally investigated eosinophils' proteoglycan (PG) expression patterns by real-time PCR, which revealed the highest expression level for serglycin. Including an anti-serglycin antibody in CCL26-induced eosinophil migration experiments reduced the chemotaxis of these immune cells, thereby proving the dependence of eosinophil mobilization on the proteoglycan serglycin. [ABSTRACT FROM AUTHOR]

In Chagas disease (ChD) caused by Trypanosoma cruzi, new biomarkers to predict chronic cardiac pathology are urgently needed. Previous studies in chagasic patients with mild symptomatology showed that antibodies against the immunodominant R3 epitope of sCha, a fragment of the human basic helix‐loop‐helix transcription factor like 5, correlated with cardiac pathology. To validate sCha as a biomarker and to understand the origin of anti‐sCha antibodies, we conducted a multicenter study with several cohorts of chagasic patients with severe cardiac symptomatology. We found that levels of antibodies against sCha discriminated the high risk of sudden death, indicating they could be useful for ChD prognosis. We investigated the origin of the antibodies and performed an alanine scan of the R3 epitope. We identified a minimal epitope MRQLD, and a BLAST search retrieved several T. cruzi antigens. Five of the hits had known or putative functions, of which phosphonopyruvate decarboxylase showed the highest cross‐reactivity with sCha, confirming the role of molecular mimicry in the development of anti‐sCha antibodies. Altogether, we demonstrate that the development of antibodies against sCha, which originated by molecular mimicry with T. cruzi antigens, could discriminate electrocardiographic alterations associated with a high risk of sudden death. [ABSTRACT FROM AUTHOR]

Advances in sequencing technologies have led to a rapid growth of public protein sequence databases, whereby the fraction of proteins with experimentally verified function continuously decreases. This problem is currently addressed by automated functional annotations with computational tools, which however lack the accuracy of experimental approaches and are susceptible to error propagation. Here, we present an approach that combines the efficiency of functional annotation by in silico methods with the rigor of enzyme characterization in vitro. First, a thorough experimental analysis of a representative enzyme of a group of homologues is performed which includes a focused alanine scan of the active site to determine a fingerprint of function-determining residues. In a second step, this fingerprint is used in combination with a sequence similarity network to identify putative isofunctional enzymes among the homologues. Using this approach in a proof-of-principle study, homologues of the histidinol phosphate phosphatase (HolPase) from Pseudomonas aeruginosa, many of which were annotated as phosphoserine phosphatases, were predicted to be HolPases. This functional annotation of the homologues was verified by in vitro testing of several representatives and an analysis of the occurrence of annotated HolPases in the corresponding phylogenetic groups. Moreover, the application of the same approach to the homologues of the HolPase from the archaeon Nitrosopumilus maritimus, which is not related to the HolPase from P. aeruginosa and was newly discovered in the course of this work, led to the annotation of the putative HolPase from various archaeal species., (© 2024 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

Electrostatic interactions are ubiquitous in proteins and dictate stability and function. In this review, we discuss several methods for the analysis of electrostatics in protein-protein interactions. We discuss alanine-scanning mutagenesis, Poisson-Boltzmann electrostatics, free energy calculations, electrostatic similarity distances, and hierarchical clustering of electrostatic potentials. Our recently developed computational framework, known as Analysis of Electrostatic Similarities Of Proteins (AESOP), incorporates these tools to efficiently elucidate the role of electrostatic potentials in protein interactions. We present the application of AESOP to several proteins and protein complexes, for which charge is purported to facilitate protein association. Specifically, we illustrate how recent work has shaped the formulation of electrostatic calculations, the correlation of electrostatic free energies and electrostatic potential clustering results with experimental binding and activity data, the pH dependence of protein stability and association, the design of mutant proteins with enhanced immunological activity, and how AESOP can expose deficiencies in structural models and experimental data. This integrative approach can be utilized to develop mechanistic models and to guide experimental studies by predicting mutations with desired physicochemical properties and function. Alteration of the electrostatic properties of proteins offers a basis for the design of proteins with optimized binding and activity.

Cyclic lipo(depsi)peptides (CLiPs) from Pseudomonas constitute a class of natural products involved in a broad range of biological functions for their producers. They also display interesting antimicrobial potential including activity against Gram-positive bacteria. Literature has indicated that these compounds can induce membrane permeabilization, possibly through pore-formation, leading to the general view that the cellular membrane constitutes the primary target in their mode of action. In support of this view, we previously demonstrated that the enantiomer of pseudodesmin A, a member of the viscosin group of CLiPs, shows identical activity against a test panel of six Gram-positive bacterial strains. Here, a previously developed total organic synthesis route is used and partly adapted to generate 20 novel pseudodesmin A analogs in an effort to derive links between molecular constitution, structure and activity. From these, the importance of a macrocycle closed by an ester bond as well as a critical length of β-OH fatty acid chain capping the N-terminus is conclusively demonstrated, providing further evidence for the importance of peptide-membrane interactions in the mode of action. Moreover, an alanine scan is used to unearth the contribution of specific amino acid residues to biological activity. Subsequent interpretation in terms of a structural model describing the location and orientation of pseudodesmin A in a membrane environment, allows first insight in the peptide-membrane interactions involved. The biological screening also identified residue positions that appear less sensitive to conservative modifications, allowing the introduction of a non-perturbing tryptophan residue which will pave the way toward biophysical studies using fluorescence spectroscopy.

We recently identified the glioblastoma homing peptide CooP (CGLSGLGVA) using in vivo phage display screen. The mammary-derived growth inhibitor (MDGI/FABP3) was identified as its interacting partner. Here, we present an alanine scan of A-CooP to investigate the contribution of each amino acid residue to the binding to FABP3 by microscale thermophoresis (MST) and surface plasmon resonance (SPR). We also tested the binding affinity of the A-CooP-K, KA-CooP, and retro-inverso A-CooP analogues to the recombinant FABP3. According to the MST analysis, A-CooP showed micromolar (KD = 2.18 µM) affinity to FABP3. Alanine replacement of most of the amino acids did not affect peptide affinity to FABP3. The A-CooP-K variant showed superior binding affinity, while A-[Ala5]CooP and A-[Ala7]CooP, both replacing a glycine residue with alanine, showed negligible binding to FABP3. These results were corroborated in vitro and in vivo using glioblastoma models. Both A-CooP-K and A-CooP showed excellent binding in vitro and homing in vivo, while A-[Ala5]CooP and control peptides failed to bind the cells or home to the intracranial glioblastoma xenografts. These results provide insight into the FABP3–A-CooP interaction that may be important for future applications of drug conjugate design and development.

The iron-sulfur (Fe/S) clusters are the most ancient co-factors of proteins involved in the most essential processes in bacterial systems and yeast, such as Saccharomyces cerevisiae. The main protein involved in the Fe/S cluster transfer is the Iron sulfur cluster assembly protein 1 (Isu1), which interacts with Jac1 during one of the stages of the Fe/S cluster biogenesis cycle forming a binary complex. In this work, the interaction interface of Isu1 was investigated by selective substitutions of amino-acid residues to understand their role in binding to the Jac1 protein. An initial alanine scan was done to limit the number of possible residues subjected to the replacement and to confirm the previously obtained results. Then, MD simulations using the coarse-grained UNRES force field were run for two selected mutants: L63V72F94 and L63V64G65D71. The analysis of the dynamics and interaction patterns of the Isu1-Jac1 complexes confirmed that the investigated residues played an important role in their binding.

The outburst of microbial resistance to antibiotics creates the need for new sources of active compounds for the treatment of pathogenic microorganisms. Marine microalgae are of particular interest in this context because they have developed tolerance and defense strategies to resist the exposure to pathogenic bacteria, viruses, and fungi in the aquatic environment. Although antimicrobial activities have been reported for some microalgae, natural algal bioactive peptides have not been described yet. In this work, acid extracts from the microalga Tetraselmis suecica with antibacterial activity were analyzed, and de novo sequences of peptides were determined. Synthetic peptides and their alanine and lysine analogs allowed identifying key residues and increasing their antibacterial activity. Additionally, it was determined that the localization of positive charges within the peptide sequence influences the secondary structure with tendency to form an alpha helical structure.

To date structure-activity relationship (SAR) studies of the dynorphins (Dyn), endogenous peptides for kappa opioid receptors (KOR), have focused almost exclusively on Dyn A with minimal studies on Dyn B. While both Dyn A and Dyn B have identical N-terminal sequences, their C-terminal sequences differ, which could result in differences in pharmacological activity. We performed an alanine scan of the non-glycine residues up through residue 11 of Dyn B amide to explore the roles of these side chains in the activity of Dyn B. The analogs were synthesized by fluorenylmethyloxycarbonyl (Fmoc)-based solid phase peptide synthesis and evaluated for their opioid receptor affinities and opioid potency and efficacy at KOR. Similar to Dyn A the N-terminal Tyr1 and Phe4 residues of Dyn B amide are critical for opioid receptor affinity and KOR agonist potency. The basic residues Arg6 and Arg7 contribute to the KOR affinity and agonist potency of Dyn B amide, while Lys10 contributes to the opioid receptor affinity, but not KOR agonist potency, of this peptide. Comparison to the Ala analogs of Dyn A (1-13) suggests that the basic residues in the C-terminus of both peptides contribute to KOR binding, but differences in their relative positions may contribute to the different pharmacological profiles of Dyn A and Dyn B. The other unique C-terminal residues in Dyn B amide also appear to influence the relative affinity of this peptide for KOR versus mu and delta opioid receptors. This SAR information may be applied in the design of new Dyn B analogs that could be useful pharmacological tools. [ABSTRACT FROM AUTHOR]

Enteroviruses are responsible for a multitude of human diseases. Expansion of the virus capsid is associated with a cascade of conformational changes that allow the subsequent release of RNA. For the first time, this study presents a comprehensive bioinformatic screen for the prediction of interacting motifs within intraprotomer interfaces and across respective interfaces surrounding the fivefold and twofold axes. The results identify a network of conserved motif residues involved in interactions in enteroviruses that may be critical to capsid stabilisation, providing guidelines towards developing antivirals that interfere with viral expansion during RNA release. [ABSTRACT FROM AUTHOR]

Monoclonal antibodies have become an important class of therapeutics in the last 30 years. Because the mechanism of action of therapeutic antibodies is intimately linked to their binding epitopes, identification of the epitope of an antibody to the antigen plays a central role during antibody drug development. The gold standard of epitope mapping, X-ray crystallography, requires a high degree of proficiency with no guarantee of success. Here, we evaluated six widely used alternative methods for epitope identification (peptide array, alanine scan, domain exchange, hydrogen-deuterium exchange, chemical cross-linking, and hydroxyl radical footprinting) in five antibody-antigen combinations (pembrolizumab+PD1, nivolumab+PD1, ipilimumab+CTLA4, tremelimumab+CTLA4, and MK-5890+CD27). The advantages and disadvantages of each technique are demonstrated by our data and practical advice on when and how to apply specific epitope mapping techniques during the drug development process is provided. Our results suggest chemical cross-linking most accurately identifies the epitope as defined by crystallography.

The short membrane-active peptide BP100 [KKLFKKILKYL-NH 2 ] is known as an effective antimicrobial and cell penetrating agent. For a functional alanine scan each of the 11 amino acids was replaced with deuterated Ala-d 3 , one at a time. MIC assays showed that a substitution of Lys did not affect the antimicrobial activity, but it decreased when a hydrophobic residue was replaced. In most cases, a reduction in hydrophobicity led to a decrease in hemolysis, and some peptide analogues had an improved therapeutic index. Circular dichroism showed that BP100 folds as an amphiphilic α-helix in a bilayer. Its alignment was determined from 2 H NMR in oriented membranes of different composition. The azimuthal rotation angle was the same under all conditions, but the average helix tilt angle and the dynamical behavior of the peptide varied in a systematic manner. In POPC/POPG bilayers, with a negative spontaneous curvature, the peptide was found to lie flat on the bilayer surface, and with little wobble. In DMPC/DMPG, with a positive spontaneous curvature, BP100 at higher concentrations became tilted obliquely into the membrane, with the uncharged C-terminus inserted more deeply into the lipid bilayer, experiencing significant fluctuations in tilt angle. In DMPC/DMPG/lyso-MPC, with a pronounced positive spontaneous curvature, the helix tilted even further and became even more mobile. The 11-mer BP100 is obviously too short to form transmembrane pores. We conclude that BP100 operates via a carpet mechanism, whereby the C-terminus gets inserted into the hydrophobic core of the bilayer, which leads to membrane perturbation and induces transient permeability. [ABSTRACT FROM AUTHOR]

An important task of biomolecular simulation is the calculation of relative binding free energies upon chemical modification of partner molecules in a biomolecular complex. The potential of mean force (PMF) along a reaction coordinate for association or dissociation of the complex can be used to estimate binding affinities. A free energy perturbation approach, termed umbrella sampling (US) perturbation, has been designed that allows an efficient calculation of the change of the PMF upon modification of a binding partner based on the trajectories obtained for the wild type reference complex. The approach was tested on the interaction of modified water molecules in aqueous solution and applied to in silico alanine scanning of a peptide-protein complex. For the water interaction test case, excellent agreement with an explicit PMF calculation for each modification was obtained as long as no long range electrostatic perturbations were considered. For the alanine scanning, the experimentally determined ranking and binding affinity changes upon alanine substitutions could be reproduced within 0.1-2.0 kcal/mol. In addition, good agreement with explicitly calculated PMFs was obtained mostly within the sampling uncertainty. The combined US and perturbation approach yields, under the condition of sufficiently small system modifications, rigorously derived changes in free energy and is applicable to any PMF calculation. [ABSTRACT FROM AUTHOR]

The AVR9 peptide of Cladosporium fulvum is an elicitor of the hypersensitive response in tomato plants carrying the Cf-9 resistance gene (MM-Cf9). To determine the structure-activity relationship of the AVR9 peptide, amino acids important for AVR9 elicitor activity were identified by independently substituting each amino acid of AVR9 by alanine. In addition, surface-exposed amino acid residues of AVR9 were substituted by other amino acids. Activity of the mutant Avr9 constructs was studied by expressing the constructs in MM-Cf9 tomato plants, using the potato virus X (PVX) expression system and assessing the severity of necrosis induced by each PVX∷Avr9 construct. This allowed direct identification of amino acid residues of AVR9 that are essential for elicitor activity. We identified amino acid substitutions that resulted in AVR9 mutants with higher, similar, or lower elicitor activity compared to the wild-type AVR9 peptide. Some mutants had completely lost elicitor activity. A selection of peptides, representing different categories, was isolated and injected into leaves of MM-Cf9 plants. The necrosis-inducing activity of the isolated peptides correlated well with the necrosis induced by the corresponding PVX∷Avr9 derivatives. Based on the necrosis-inducing activity of the mutant AVR9 peptides and the global structure of AVR9, we assigned sites in AVR9 that are important for its necrosis-inducing activity. We postulate that the “hydrophobic β-loop” region of the AVR9 peptide is crucial for necrosis-inducing activity in tomato plants that carry the Cf-9 resistance gene.

Proteasome is a 'proteolytic factory' that constitutes an essential part of the ubiquitin-proteasome pathway. The involvement of proteasome in regulation of all major aspects of cellular physiology makes it an attractive drug target. So far, only inhibitors of the proteasome entered the clinic as anti-cancer drugs. However, proteasome regulators may also be useful for treatment of inflammatory and neurodegenerative diseases. We established in our previous studies that the peptide Tat2, comprising the basic domain of HIV-1 Tat protein: R49KKRRQRR56, supplemented with Q66DPI69 fragment, inhibits the 20S proteasome in a noncompetitive manner. Mechanism of Tat2 likely involves allosteric regulation because it competes with the proteasome natural 11S activator for binding to the enzyme noncatalytic subunits. In this study, we performed alanine walking coupled with biological activity measurements and FTIR and CD spectroscopy to dissect contribution of a charge and conformation of Tat2 to its capability to influence peptidase activity of the proteasome. In solution, Tat2 and most of its analogs with a single Ala substitution preferentially adopted a conformation containing PPII/turn structural motifs. Replacing either Asp10 or two or more adjacent Arg/Lys residues induced a random coil conformation, probably by disrupting ionic interactions responsible for stabilization of the peptides ordered structure. The random coil Tat2 analogs lost their capability to activate the latent 20S proteasome. In contrast, inhibitory properties of the peptides more significantly depended on their positive charge. The data provide valuable clues for the future optimization of the Tat2-based proteasome regulators. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

The CGRP (calcitonin gene-related peptide) receptor is a family B GPCR (G-protein-coupled receptor). It consists of a GPCR, CLR (calcitonin receptor-like receptor) and an accessory protein, RAMP1 (receptor activity modifying protein 1). RAMP1 is needed for CGRP binding and also cell-surface expression of CLR. CLR is an example of a family B GPCR. Unlike family A GPCRs, little is known about how these receptors are activated by their endogenous ligands. This review considers what is known about the activation of family B GPCRs and then considers how this might be applied to CLR, particularly in light of new knowledge of the crystal structures of family A GPCRs. [ABSTRACT FROM AUTHOR]

Core peptide is a hydrophobic peptide, the sequence of which is derived from the T-cell antigen receptor alpha-chain transmembrane region. Previous studies have shown that core peptide can inhibit T-cell-mediated immune responses both in vitro and in vivo. Here, we report the role each constituent amino acid plays within core peptide using an alanine scan and the amino acid effect on function using a biological antigen presentation assay. The biophysical behaviour of these analogues in model membranes was analysed using surface plasmon resonance studies and then binding correlated with T-cell function. Removal of any single hydrophobic amino acid between the two charged amino acids in core peptide (R, K) resulted in lower binding. Changing the overall net charge of core peptide, by removing either of the positively charged residues (R or K), had varying effects on peptide binding and IL-2 production. There was a direct correlation (ρ = 0.718) between peptide binding to model membranes and peptide ability to inhibit IL-2. Except for IL-2 inhibition, production of other T-cell cytokines such as GM-CSF, IFN-γ, IL-1α, IL-4, IL-5, IL-6, IL-10, IL-17 and T-cell antigen receptor alpha-chain was not detected using a fluorescent bead immunoassay. This study provides important structure-function relationships essential for further drug design. [ABSTRACT FROM AUTHOR]

Abstract: Feglymycin (FGM), a natural Streptomyces-derived 13mer peptide, consistently inhibits HIV replication in the lower μM range. FGM also inhibits HIV cell-to-cell transfer between HIV-infected T cells and uninfected CD4+ T cells and the DC-SIGN-mediated viral transfer to CD4+ T cells. FGM potently interacts with gp120 (X4 and R5) as determined by SPR analysis and shown to act as a gp120/CD4 binding inhibitor. Alanine-scan analysis showed an important role for l-aspartic acid at position 13 for its anti-HIV activity. In vitro generated FGM-resistant HIV-1 IIIB virus (HIV-1 IIIBFGMres) showed two unique mutations in gp120 at positions I153L and K457I. HIV-1 IIIBFGMres virus was equally susceptible to other viral binding/adsorption inhibitors with the exception of dextran sulfate (9-fold resistance) and cyclotriazadisulfonamide (>15-fold), two well-described compounds that interfere with HIV entry. In conclusion, FGM is a unique prototype lead peptide with potential for further development of more potent anti-HIV derivatives. [Copyright &y& Elsevier]

Virulence factors EfbC and Ehp from Staphylococcus aureus are potent inhibitors of complement activation. Both are excessively charged and bind to complement protein C3d at an acidic interface. We computationally generated single-alanine mutants of charged residues in the C3d-EfbC and C3d-Ehp complexes, and utilized electrostatic clustering and Poisson-Boltzmann free energy calculations to evaluate the role of electrostatics in association. Our results indicate that both interfacial electrostatic interactions and electrostatic potential distribution are crucial for C3d-EfbC and C3d-Ehp association. The results presented herein serve as a predictive tool in the selection of mutants with desired binding and immunological activity, and will narrow the search for viable candidates for further computational and experimental analyses. This study serves as the foundation for development of an inhibitor of the C3d-EfbC and C3d-Ehp interactions to combat bacterial infection and design of a complement inhibitor using EfbC and Ehp as a model. [ABSTRACT FROM AUTHOR]

Abstract: The HIV-1 integrase protein (IN) mediates integration of the viral cDNA into the host genome and is a target for anti-HIV drugs. We have recently described a peptide derived from residues 361–370 of the IN cellular partner protein LEDGF/p75, which inhibited IN catalytic activity in vitro and HIV-1 replication in cells. Here we performed a comprehensive study of the LEDGF 361–370 mechanism of action in vitro, in cells and in vivo. Alanine scan, fluorescence anisotropy binding studies, homology modeling and NMR studies demonstrated that all residues in LEDGF 361–370 contribute to IN binding and inhibition. Kinetic studies in cells showed that LEDGF 361–370 specifically inhibited integration of viral cDNA. Thus, the full peptide was chosen for in vivo studies, in which it inhibited the production of HIV-1 RNA in mouse model. We conclude that the full LEDGF 361–370 peptide is a potent HIV-1 inhibitor and may be used for further development as an anti-HIV lead compound. [Copyright &y& Elsevier]

Abstract: The calcitonin receptor-like receptor (CLR) acts as a receptor for the calcitonin gene-related peptide (CGRP) but in order to recognize CGRP, it must form a complex with an accessory protein, receptor activity modifying protein 1 (RAMP1). Identifying the protein/protein and protein/ligand interfaces in this unusual complex would aid drug design. The role of the extreme N-terminus of CLR (Glu23-Ala60) was examined by an alanine scan and the results were interpreted with the help of a molecular model. The potency of CGRP at stimulating cAMP production was reduced at Leu41Ala, Gln45Ala, Cys48Ala and Tyr49Ala; furthermore, CGRP-induced receptor internalization at all of these receptors was also impaired. Ile32Ala, Gly35Ala and Thr37Ala all increased CGRP potency. CGRP specific binding was abolished at Leu41Ala, Ala44Leu, Cys48Ala and Tyr49Ala. There was significant impairment of cell surface expression of Gln45Ala, Cys48Ala and Tyr49Ala. Cys48 takes part in a highly conserved disulfide bond and is probably needed for correct folding of CLR. The model suggests that Gln45 and Tyr49 mediate their effects by interacting with RAMP1 whereas Leu41 and Ala44 are likely to be involved in binding CGRP. Ile32, Gly35 and Thr37 form a separate cluster of residues which modulate CGRP binding. The results from this study may be applicable to other family B GPCRs which can associate with RAMPs. [Copyright &y& Elsevier]

The protein scaffold is a peptide framework with a high tolerance of residue modifications. The cysteine‐stabilized αβ motif (CSαβ) consists of an α‐helix and an antiparallel triple‐stranded β‐sheet connected by two disulfide bridges. Proteins containing this motif share low sequence identity but high structural similarity and has been suggested as a good scaffold for protein engineering. The Vigna radiate defensin 1 (VrD1), a plant defensin, serves here as a model protein to probe the amino acid tolerance of CSαβ motif. A systematic alanine substitution is performed on the VrD1. The key residues governing the inhibitory function and structure stability are monitored. Thirty‐two of 46 residue positions of VrD1 are altered by site‐directed mutagenesis techniques. The circular dichroism spectrum, intrinsic fluorescence spectrum, and chemical denaturation are used to analyze the conformation and structural stability of proteins. The secondary structures were highly tolerant to the amino acid substitutions; however, the protein stabilities were varied for each mutant. Many mutants, although they maintained their conformations, altered their inhibitory function significantly. In this study, we reported the first alanine scan on the plant defensin containing the CSαβ motif. The information is valuable to the scaffold with the CSαβ motif and protein engineering. [ABSTRACT FROM AUTHOR]

Abstract: SARS-CoV entry is mediated by spike glycoprotein. During the viral and host cellular membrane fusion, HR1 and HR2 form 6-helix bundle, positioning the fusion peptide closely to the C-terminal region of ectodomain to drive apposition and subsequent membrane fusion. Connecting to the HR2 region is a Trp-rich region which is absolutely conserved in members of coronaviruses. To investigate the importance of Trp-rich region in SARS-CoV entry, we produced different mutated S proteins using Alanine scan strategy. SARS-CoV pseudotyped with mutated S protein was used to measure viral infectivity. To restore the aromaticity of Ala-mutants, we performed rescue experiments using phenylalanine substitutions. Our results show that individually substituted Ala-mutants substantially decrease infectivity by >90%, global Ala-mutants totally abrogated infectivity. In contrast, Phe-substituted mutants are able to restore 10–25% infectivity comparing to the wild-type. The results suggest that the Trp-rich region of S protein is essential for SARS-CoV infectivity. [Copyright &y& Elsevier]

Fallaxin is a 25-mer antibacterial peptide amide, which was recently isolated from the West Indian mountain chicken frog Leptodactylus fallax. Fallaxin has been shown to inhibit the growth of several Gram-negative bacteria including Enterobacter cloacae, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa. Here, we report a structure-activity study of fallaxin based on 65 analogs, including a complete alanine scan and a full set of N- and C-terminal truncated analogs. The fallaxin analogs were tested for hemolytic activity and antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-intermediate resistant S. aureus, ( VISA), methicillin-susceptible S. aureus ( MSSA), E. coli, K. pneumoniae, and P. aeruginosa. We identified several analogs, which showed improved antibacterial activity compared to fallaxin. Our best candidate was FA12, which displayed MIC values of 3.12, 25, 25, and 50 μM against E. coli, K. pneumoniae, MSSA, and VISA, respectively. Furthermore, we correlated the antibacterial activity with various structural parameters such as charge, hydrophobicity 〈H〉, mean hydrophobic moment 〈μH〉, and α-helicity. We were able to group the active and inactive analogs according to mean hydrophobicity 〈H〉 and mean hydrophobic moment 〈μH〉. Far-UV CD-spectroscopy experiments on fallaxin and several analogs in buffer, in TFE, and in membrane mimetic environments (small unilamellar vesicles) indicated that a coiled-coil conformation could be an important structural trait for antibacterial activity. This study provides data that support fallaxin analogs as promising lead structures in the development of new antibacterial agents. [ABSTRACT FROM AUTHOR]

Abstract: A region of subunit IV comprising residues 77–85 is identified as essential for interaction with the core complex to restore the bc 1 activity (reconstitutive activity). Recombinant subunit IV mutants with single or multiple alanine substitution at this region were generated and characterized to identify the essential amino acid residues. Residues 81–84, with sequence of YRYR, are required for reconstitutive activity of subunit IV, because a mutant with these four residues substituted with alanine has little activity, while a mutant with alanine substitution at residues 77–80 and 85 have the same reconstitutive activity as that of the wild-type IV. The positively charged group at R-82 and R-84 and both the hydroxyl group and aromatic group at Y-81 and Y-83 are essential. The interactions between these four residues of subunit IV and residues of core subunits are also responsible for the stability of the complex. However, these interactions are not essential for the incorporation of subunit IV into the bc 1 complex. [Copyright &y& Elsevier]

The nuclear localization signal sequence (NLS) of the transcription factor NF- κB is a cationic peptide with the ability to cross the cytoplasmic membrane and facilitate the delivery of attached cargo, such as DNA and proteins, to cells. Previous research had pointed to the essential role of cationic residues, therefore, the importance of residues within the NLS of NF- κB was evaluated for cellular uptake using an alanine replacement strategy. Although it was expected that removal of the cationic groups would have the greatest effect on membrane translocation, the most significant decreases in cellular uptake occurred with the replacement of the hydrophilic Q6 (80%) and the hydrophobic L8 (70%) residues. Replacement of the positively charged residues resulted in 30–40% decrease in cellular uptake, indicating that electrostatic interactions are not the primary driving force for membrane translocation. [ABSTRACT FROM AUTHOR]

Abstract: A nonphosphorylated disulfide-bridged peptide, cyclo(Cys-Glu[sup 1]-Leu-Tyr-Glu-Asn-Val-Gly-Met-Tyr[sup 9]-Cys)-amide (termed G1) has been identified, by phage library, that binds to the Grb2-SH2 domain but not the src SH2 domain. Synthetic G1 blocks the Grb2-SH2 domain association (IC[sub 50] of 15.5 µm) with natural phosphopeptide ligands. As a new structural motif that binds to the Grb2-SH2 domain in a pTyr-independent manner, the binding affinity of G1 is contributed by the highly favored interactions of its structural elements interacting with the binding pocket of the protein. These interactions involve side-chains of amino acids Glu[sup 1], Tyr[sup 3], Glu[sup 4], Asn[sup 5], and Met[sup 8]. Also a specific conformation is required for the cyclic peptide when bound to the protein. Ala scanning within G1 and molecular modeling analysis suggest a promising model in which G1 peptide binds in the phosphotyrosine binding site of the Grb2-SH2 domain in a β-turn-like conformation. Replacement of Tyr[sup 3] or Asn[sup 5] with Ala abrogates the inhibitory activity of the peptide, indicating that G1 requires a Y-X-N consensus sequence similar to that found in natural pTyr-containing ligands, but without Tyr phosphorylation. Significantly, the Ala mutant of Glu[sup 1], i.e. the amino acid N-terminal to Y[sup 3], remarkably reduces the binding affinity. The position of the Glu[sup 1] side-chain is confirmed to provide a complementary role for pTyr[sup 3], as demonstrated by the low micromolar inhibitory activity (IC[sub 50] = 1.02 µm) of the nonphosphorylated peptide 11, G1(Gla[sup 1]), in which Glu[sup 1] was replaced by γ-carboxy-glutamic acid (Gla). [ABSTRACT FROM AUTHOR]

WAP-8294A2 is a cyclic peptide antibiotic with novel structure and excellent activity against Gram-positive pathogens. Herein, we report the total synthesis of complex macrocyclic peptide WAP-8294A2 (W 1) , ent -analogue W 2 , deoxy analogue W 3 and de-methyl analogue W 4 using a solid-phase synthetic route followed by a final stage solution-phase cyclization reaction. Exploitation of this process allowed the synthesis of eleven alanine-scanning analogues and eight lysine-scanning analogues. The antimicrobial activity of these analogues was evaluated in vitro against Gram-positive bacteria. Based on the MIC results, a primary systematic structure-activity relationship has been established. [ABSTRACT FROM AUTHOR]

We recently identified the glioblastoma homing peptide CooP (CGLSGLGVA) using in vivo phage display screen. The mammary-derived growth inhibitor (MDGI/FABP3) was identified as its interacting partner. Here, we present an alanine scan of A-CooP to investigate the contribution of each amino acid residue to the binding to FABP3 by microscale thermophoresis (MST) and surface plasmon resonance (SPR). We also tested the binding affinity of the A-CooP-K, KA-CooP, and retro-inverso A-CooP analogues to the recombinant FABP3. According to the MST analysis, A-CooP showed micromolar (KD = 2.18 µM) affinity to FABP3. Alanine replacement of most of the amino acids did not affect peptide affinity to FABP3. The A-CooP-K variant showed superior binding affinity, while A-[Ala5]CooP and A-[Ala7]CooP, both replacing a glycine residue with alanine, showed negligible binding to FABP3. These results were corroborated in vitro and in vivo using glioblastoma models. Both A-CooP-K and A-CooP showed excellent binding in vitro and homing in vivo, while A-[Ala5]CooP and control peptides failed to bind the cells or home to the intracranial glioblastoma xenografts. These results provide insight into the FABP3–A-CooP interaction that may be important for future applications of drug conjugate design and development. [ABSTRACT FROM AUTHOR]

There is interest in peptide drug design, especially for targeting intracellular protein–protein interactions. Therefore, the experimental validation of a computational platform for enabling peptide drug design is of interest. Here, we describe our peptide drug design platform (CMDInventus) and demonstrate its use in modeling and predicting the structural and binding aspects of diverse peptides that interact with oncology targets MDM2/MDMX in comparison to both retrospective (pre-prediction) and prospective (post-prediction) data. In the retrospective study, CMDInventus modules (CMDpeptide, CMDboltzmann, CMDescore and CMDyscore) were used to accurately reproduce structural and binding data across multiple MDM2/MDMX data sets. In the prospective study, CMDescore, CMDyscore and CMDboltzmann were used to accurately predict binding affinities for an Ala-scan of the stapled α-helical peptide ATSP-7041. Remarkably, CMDboltzmann was used to accurately predict the results of a novel D-amino acid scan of ATSP-7041. Our investigations rigorously validate CMDInventus and support its utility for enabling peptide drug design. [ABSTRACT FROM AUTHOR]

Cyclic lipo(depsi)peptides (CLiPs) from Pseudomonas constitute a class of natural products involved in a broad range of biological functions for their producers. They also display interesting antimicrobial potential including activity against Gram-positive bacteria. Literature has indicated that these compounds can induce membrane permeabilization, possibly through pore-formation, leading to the general view that the cellular membrane constitutes the primary target in their mode of action. In support of this view, we previously demonstrated that the enantiomer of pseudodesmin A, a member of the viscosin group of CLiPs, shows identical activity against a test panel of six Gram-positive bacterial strains. Here, a previously developed total organic synthesis route is used and partly adapted to generate 20 novel pseudodesmin A analogs in an effort to derive links between molecular constitution, structure and activity. From these, the importance of a macrocycle closed by an ester bond as well as a critical length of β-OH fatty acid chain capping the N-terminus is conclusively demonstrated, providing further evidence for the importance of peptide-membrane interactions in the mode of action. Moreover, an alanine scan is used to unearth the contribution of specific amino acid residues to biological activity. Subsequent interpretation in terms of a structural model describing the location and orientation of pseudodesmin A in a membrane environment, allows first insight in the peptide-membrane interactions involved. The biological screening also identified residue positions that appear less sensitive to conservative modifications, allowing the introduction of a non-perturbing tryptophan residue which will pave the way toward biophysical studies using fluorescence spectroscopy., (Copyright © 2020 De Vleeschouwer, Van Kersavond, Verleysen, Sinnaeve, Coenye, Martins and Madder.)