Peptide Binding Research Articles

Investigating Cellulose Binding of Peptides Derived from Carbohydrate Binding Module 1.

Carbohydrate-binding modules (CBM) have emerged as useful tools for a wide range of tasks, including the use as purification tags or for cellulose fiber modification. For this purpose, the CBM needs to be attached to a target protein leading to large constructs. We investigated if short peptides from the carbohydrate binding site of CBMs can bind in a similar way as native, full-length CBMs to nanocrystalline cellulose (NCC) or cotton linter paper. We designed our cellulose-binding peptides to be less hydrophobic and shorter than those previously reported. Starting from the binding site of Cel7A-CBM1, we incorporated the essential amino acids involved in cellulose binding into our peptides. These peptides, as well as control peptides with scrambled sequences or a lack of essential amino acids, bound to cellulose with similar affinity as CBM regardless of their secondary structure, sequence, or hydrophobicity. This unspecific mode of cellulose binding displayed by the presented peptides may be exploited to functionalize cellulose-based biomaterials by means of peptide-conjugates.

Site-Selective Functionalization of Molecularly Imprinted Nanoparticles to Recognize Lysine-Rich Peptides.

Sequence-selective binding of peptides has been a long-standing goal of chemists. As one of the most abundant amino acids in proteins, lysine plays an important role in protein functions as well as in antimicrobial and cell-penetrating peptides. Herein, we report molecularly imprinted nanoparticles (NPs) with high sequence selectivity for lysine-rich peptides. The NPs are prepared from molecular imprinting of cross-linkable surfactant micelles and postmodification of the imprinted pockets by photoaffinity labeling. The method allows carboxylic acids to be installed precisely near the lysine amino side chains, greatly enhancing the binding strengths of lysine-rich peptides. Small variations in the peptide sequence can be distinguished, and the binding affinity correlates positively with the number of lysine groups in model tripeptides. The method applies to complex lysine-rich biological peptides, achieving hundreds of nanomolar binding affinities and excellent binding specificities.

MoPPIt: De Novo Generation of Motif-Specific Binders with Protein Language Models.

The ability to precisely target specific motifs on disease-related proteins, whether conserved epitopes on viral proteins, intrinsically disordered regions within transcription factors, or breakpoint junctions in fusion oncoproteins, is essential for modulating their function while minimizing off-target effects. Current methods struggle to achieve this specificity without reliable structural information. In this work, we introduce a motif-specific PPI targeting algorithm, moPPIt, for de novo generation of motif-specific peptide binders from the target protein sequence alone. At the core of moPPIt is BindEvaluator, a transformer-based model that interpolates protein language model embeddings of two proteins via a series of multi-headed self-attention blocks, with a key focus on local motif features. Trained on over 510,000 annotated PPIs, BindEvaluator accurately predicts target binding sites given protein-protein sequence pairs with a test AUC > 0.94, improving to AUC > 0.96 when fine-tuned on peptide-protein pairs. By combining BindEvaluator with our PepMLM peptide generator and genetic algorithm-based optimization, moPPIt generates peptides that bind specifically to user-defined residues on target proteins. We demonstrate moPPIt's efficacy in computationally designing binders to specific motifs, first on targets with known binding peptides and then extending to structured and disordered targets with no known binders. In total, moPPIt serves as a powerful tool for developing highly specific peptide therapeutics without relying on target structure or structure-dependent latent spaces.

Competitive Interaction of the SGFRKMAF Peptide with 3CLpro Dimerization Intermediates: A Brownian Dynamics Investigation.

The SGFRKMAF peptide is known to inhibit the dimerization of 3CLpro monomers, which is essential for SARS-CoV-2 replication. The mechanism behind this, however, is largely unknown. In this work, we used Brownian dynamics simulations to compare and contrast 3CLpro monomer-monomer interactions and 3CLpro monomer-SGFRKMAF peptide interactions. We found that formation of the 3CLpro wild-type dimer could potentially involve formation of three intermediates that are primarily stabilized by G11-G124, S1-S301, and T118-G278 interactions. Analysis of 3CLpro monomer interaction with the SGFRKMAF peptide, however, revealed the presence of eight basins of interactions where the peptide assumes the highest local densities at the 3CLPro monomer surface. The second highest-density basin was found to coincide with the interface region of the wild-type 3CLpro dimer, thereby directly blocking the 3CLpro dimer-dimer interactions. The other basins, however, were found to lie far from the interface region. Notably, we found that only 6% of the BD trajectories end up directly into the basin at the interface region and ∼39% of the trajectories end up into those basins lying away from the interface region, indicating a greater role for peptide binding at sites away from the dimer interface region. Importantly, the locations of the basins lying away from the interface were found to coincide with the 3CLpro residues involved in stabilization of the 3CLpro monomer-monomer intermediates. Given that the rate constant of the peptide reaching the monomer surface was found to be almost an order of magnitude higher than the rate constant of monomer-monomer association, the SGFRKMAF peptide has the potential to inhibit dimerization of 3CLpro monomers not only through blocking the interface region but also through blocking the formation of the intermediates involved in the dimerization process. This could potentially open new avenues for 3CLpro dimerization inhibitors that transcend traditional X-ray-based discovery approaches.

PepBinding: A Workflow for Predicting Peptide Binding Structures by Combining Peptide Docking and Peptide Gaussian Accelerated Molecular Dynamics Simulations.

Predicting protein-peptide interactions is crucial for understanding peptide binding processes and designing peptide drugs. However, traditional computational modeling approaches face challenges in accurately predicting peptide-protein binding structures due to the slow dynamics and high flexibility of the peptides. Here, we introduce a new workflow termed "PepBinding" for predicting peptide binding structures, which combines peptide docking, all-atom enhanced sampling simulations using the Peptide Gaussian accelerated Molecular Dynamics (Pep-GaMD) method, and structural clustering. PepBinding has been demonstrated on seven distinct model peptides. In peptide docking using HPEPDOCK, the peptide backbone root-mean-square deviations (RMSDs) of their bound conformations relative to X-ray structures ranged from 3.8 to 16.0 Å, corresponding to the medium to inaccurate quality models according to the Critical Assessment of PRediction of Interactions (CAPRI) criteria. The Pep-GaMD simulations performed for only 200 ns significantly improved the docking models, resulting in five medium and two acceptable quality models. Therefore, PepBinding is an efficient workflow for predicting peptide binding structures and is publicly available at https://github.com/MiaoLab20/PepBinding.

Targeting Cardiac Resident CCR2+ Macrophage-Secreted MCP-1 to Attenuate Inflammation After Myocardial Infarction

After myocardial infarction (MI), cardiac resident CCR2+ macrophages release various cytokines and chemokines, notably monocyte chemoattractant protein-1 (MCP-1). MCP-1 is instrumental in recruiting CCR2+ monocytes to the damaged region. The excessive arrival of these monocytes, which then become macrophages, perpetuates inflammation at the site of injury. This continuous inflammation leads to adverse tissue remodeling and compromises cardiac function over time. We hypothesized that neutralizing the MCP-1 secreted by cardiac resident CCR2+ macrophages can mitigate post-MI inflammation by curtailing the recruitment of monocytes and their differentiation into macrophages. In this work, we developed nanoparticles that target the infarcted heart, specifically accumulating in the damaged area after intravenous (IV) administration, and docking onto CCR2+ macrophages. These nanoparticles were designed to slowly release an MCP-1 binding peptide, HSWRHFHTLGGG (HSW), which neutralizes the upregulated MCP-1. We showed that the HSW reduced monocyte migration, inhibited pro-inflammatory cytokine upregulation, and suppressed myofibroblast differentiation in vitro. After IV delivery, the released HSW significantly decreased monocyte recruitment and pro-inflammatory macrophage density, increased cardiac cell survival, attenuated cardiac fibrosis, and improved cardiac function. Taken together, our findings support the strategy of MCP-1 neutralization at the acute phase of MI as a promising way to alleviate post-MI inflammation. STATEMENT OF SIGNIFICANCE: After a myocardial infarction (MI), CCR2+ macrophages resident in the heart release various cytokines and chemokines, notably monocyte chemoattractant protein-1 (MCP-1). MCP-1 is instrumental in attracting CCR2+ monocytes to the damaged region. The excessive arrival of these monocytes, which then become macrophages, perpetuates inflammation at the site of injury. This continuous inflammation leads to adverse tissue remodeling and compromises cardiac function over time. In this work, we tested the hypothesis that neutralizing the MCP-1 secreted by cardiac CCR2+ macrophages can mitigate post-MI inflammation by curtailing the recruitment of monocytes.

The influence of HLA genetic variation on plasma protein expression

Genetic variation in the human leukocyte antigen (HLA) loci is associated with risk of immune-mediated diseases, but the molecular effects of HLA polymorphism are unclear. Here we examined the effects of HLA genetic variation on the expression of 2940 plasma proteins across 45,330 Europeans in the UK Biobank, with replication analyses across multiple ancestry groups. We detected 504 proteins affected by HLA variants (HLA-pQTL), including widespread trans effects by autoimmune disease risk alleles. More than 80% of the HLA-pQTL fine-mapped to amino acid positions in the peptide binding groove. HLA-I and II affected proteins expressed in similar cell types but in different pathways of both adaptive and innate immunity. Finally, we investigated potential HLA-pQTL effects on disease by integrating HLA-pQTL with fine-mapped HLA-disease signals in the UK Biobank. Our data reveal the diverse effects of HLA genetic variation and aid the interpretation of associations between HLA alleles and immune-mediated diseases.

Transient Structural Properties of the Rho GDP-Dissociation Inhibitor.

Rho GTPases, master spatial regulators of a wide range of cellular processes, are orchestrated by complex formation with guanine nucleotide dissociation inhibitors (RhoGDIs). These have been thought to possess an unstructured N-terminus that inhibits nucleotide exchange of their client upon binding/folding. Via NMR analyses, molecular dynamics simulations, and biochemical assays, we reveal instead pertinent structural properties transiently maintained both, in the presence and absence of the client, imposed onto the terminus context-specifically by modulating interactions with the surface of the folded C-terminal domain. These observations revise the long-standing textbook picture of the GTPases' mechanism of membrane extraction. Rather than by a disorder-to-order transition upon binding of an inhibitory peptide, the intricate and highly selective extraction process of RhoGTPases is orchestrated via a dynamic ensemble bearing preformed transient structural properties, suitably modulated by the specific surrounding along the multi-step process.

Transiente Strukturelle Eigenschaften des Rho‐GDP‐Dissoziationsinhibitors

AbstractRho‐GTPasen, räumliche Hauptregulatoren einer Vielzahl zellulärer Prozesse, werden durch Komplexbildung mit Guaninnukleotid‐Dissoziationsinhibitoren (RhoGDIs) reguliert. Man nimmt bisher an, dass diese einen unstrukturierten N‐Terminus aufweisen, der den Nukleotidaustausch ihres Klienten bei der Bindung/Faltung hemmt. Mithilfe von NMR‐Analysen, Molekulardynamiksimulationen und biochemischen Tests konnten wir stattdessen relevante strukturelle Eigenschaften des Terminus nachweisen, die sowohl in Anwesenheit als auch in Abwesenheit des Klienten transient existieren, allerdings kontextspezifisch variiert werden. Diese Beobachtungen revidieren das seit Langem bestehende Bild zum Mechanismus der Membranextraktion der GTPasen. Der komplexe und hochselektive Extraktionsprozess der RhoGTPasen wird nicht durch das gleichzeitige Falten und Binden eines sterisch blockierenden Peptides, sondern durch ein dynamisches Ensemble mit vordefinierten transienten strukturellen Eigenschaften orchestriert, die durch die spezifische Umgebung entlang des mehrstufigen Prozesses noch moduliert werden können.

Ligand Gaussian Accelerated Molecular Dynamics 3 (LiGaMD3): Improved Calculations of Binding Thermodynamics and Kinetics of Both Small Molecules and Flexible Peptides.

Binding thermodynamics and kinetics play critical roles in drug design. However, it has proven challenging to efficiently predict ligand binding thermodynamics and kinetics of small molecules and flexible peptides using conventional molecular dynamics (cMD), due to limited simulation time scales. Based on our previously developed ligand Gaussian accelerated molecular dynamics (LiGaMD) method, we present a new approach, termed "LiGaMD3″, in which we introduce triple boosts into three individual energy terms that play important roles in small-molecule/peptide dissociation, rebinding, and system conformational changes to improve the sampling efficiency of small-molecule/peptide interactions with target proteins. To validate the performance of LiGaMD3, MDM2 bound by a small molecule (Nutlin 3) and two highly flexible peptides (PMI and P53) were chosen as the model systems. LiGaMD3 could efficiently capture repetitive small-molecule/peptide dissociation and binding events within 2 μs simulations. The predicted binding kinetic constant rates and free energies from LiGaMD3 were in agreement with the available experimental values and previous simulation results. Therefore, LiGaMD3 provides a more general and efficient approach to capture dissociation and binding of both small-molecule ligands and flexible peptides, allowing for accurate prediction of their binding thermodynamics and kinetics.

Palmitoylation enhances short polar peptide permeation across stratum corneum lipid bilayer: A molecular dynamics study

Designing chemical molecules that target the skin for non-invasive transdermal drug delivery is of significant interest for both wound healing and skincare applications. These skin-targeting molecules must permeate the outermost protective layer of the skin, the stratum corneum (SC), which consists of dead corneocytes embedded in a lipid matrix, to fulfill their biological functions. Adsorption onto and diffusion through the lipid matrix in the SC represent two key steps for the successful permeation of a skin-targeting molecule across the SC into the underlying skin layers. Here we compare the effects of cyclization and palmitoylation on the adsorption and diffusion of a short polar peptide across a model SC lipid bilayer using molecular dynamics simulations. The cyclized peptide showed slightly better binding to the SC lipid bilayer and similar interaction energies with SC lipids compared to the unmodified peptide. In contrast, the palmitoylated peptide exhibited much stronger interaction with SC lipids via insertion of its attached fatty acid tail into the SC lipid bilayer. The average diffusivity of the cyclized peptide across the SC lipid bilayer was approximately twice that of the unmodified peptide, whereas the palmitoylated peptide’s diffusivity was about 2.7 times higher. Thus, palmitoylation appears to be a promising strategy for enhancing the binding and permeability of short polar peptides across the SC lipid matrix.

De Novo Design of Peptide Binders to Conformationally Diverse Targets with Contrastive Language Modeling.

Designing binders to target undruggable proteins presents a formidable challenge in drug discovery, requiring innovative approaches to overcome the lack of putative binding sites. Recently, generative models have been trained to design binding proteins via three-dimensional structures of target proteins, but as a result, struggle to design binders to disordered or conformationally unstable targets. In this work, we provide a generalizable algorithmic framework to design short, target-binding linear peptides, requiring only the amino acid sequence of the target protein. To do this, we propose a process to generate naturalistic peptide candidates through Gaussian perturbation of the peptidic latent space of the ESM-2 protein language model, and subsequently screen these novel linear sequences for target-selective interaction activity via a CLIP-based contrastive learning architecture. By integrating these generative and discriminative steps, we create a Peptide Prioritization via CLIP (PepPrCLIP) pipeline and validate highly-ranked, target-specific peptides experimentally, both as inhibitory peptides and as fusions to E3 ubiquitin ligase domains, demonstrating functionally potent binding and degradation of conformationally diverse protein targets in vitro. Overall, our design strategy provides a modular toolkit for designing short binding linear peptides to any target protein without the reliance on stable and ordered tertiary structure, enabling generation of programmable modulators to undruggable and disordered proteins such as transcription factors and fusion oncoproteins.

IgLON5-IgG: Innocent Bystander or Perpetrator?

Anti-IgLON5 (IgLON5-IgG)-associated disease is a newly defined clinical entity. This literature review aims to evaluate its pathogenesis, which remains a pivotal question. Features that favour a primary neurodegenerative mechanism include the non-inflammatory tauopathy neuropathological signature and overrepresentation of microtubule-associated protein tau (MAPT) H1/H1 genotype as seen in other sporadic tauopathies. In contrast, the cell-surface localisation of IgLON5, capability of anti-IgLON5 antibodies to exert direct in vitro pathogenicity and disrupt IgLON5 interactions with its binding partners, human leukocyte antigen (HLA)-DRB1*10:01 and HLA-DQB1*05:01 allele preponderance with high affinity binding of IgLON5 peptides, and responsiveness to immunotherapy favour a primary autoimmune process. The presentation and course of anti-IgLON5-associated disease is heterogenous; hence, we hypothesise that a multitude of immune mechanisms are likely simultaneously operational in this disease cohort.

Enhanced solubility and calcium absorption of calcium gluconate and calcium citrate in the presence of aspartic acid and glutamic acid

In the present study, the effects of aspartic acid and glutamic acid on the solubility and absorption of calcium gluconate and calcium citrate, two common forms of calcium supplements, were investigated. The results revealed that both amino acids increased the solubility of calcium gluconate and calcium citrate by binary or ternary complex formation, not by ligand exchange, as confirmed by calcium specification and density function theory calculations. The same mechanism applied to the calcium binding peptides. Furthermore, the addition of aspartic acid and glutamic acid resulted in a remarkable increase in calcium absorption compared to calcium gluconate and calcium citrate alone. This might be due to the altered properties (solubility, stability, and charges) of the complexes, as well as their enhanced interaction with the calcium channel TRPV6 through hydrogen bonding. Our findings provide new insights into the development of effective calcium supplementation strategies based on calcium-amino acids/calcium binding peptides complexes.

Rational design of glycosaminoglycan binding cyclic peptides using cPEPmatch

Cyclic peptides present a robust platform for drug design, offering high specificity and stability due to their conformationally constrained structures. In this study, we introduce an updated version of the Cyclic Peptide Matching program (cPEPmatch) tailored for the identification of cyclic peptides capable of mimicking protein-glycosaminoglycan (GAG) binding sites. We focused on engineering cyclic peptides to replicate the GAG-binding affinity of antithrombin III (ATIII), a protein that plays a crucial role in modulating anticoagulation through interaction with the GAG heparin. By integrating computational and experimental methods, we successfully identified a cyclic peptide binder with promising potential for future optimization. MD simulations and MM-GBSA calculations were used to assess binding efficacy, supplemented by umbrella sampling to approximate free energy landscapes. The binding specificity was further validated through NMR and ITC experiments. Our findings demonstrate that the computationally designed cyclic peptides effectively target GAGs, suggesting their potential as novel therapeutic agents. This study advances our understanding of peptide-GAG interactions and lays the groundwork for future development of cyclic peptide-based therapeutics.

High-throughput screening for cell binding and repulsion peptides on multifunctionalized surfaces

The adhesion of cells to the extracellular matrix engages cell surface receptors such as integrins, proteoglycans and other types of cell adhesion molecules such as CD44. To closely examine the determinants of cell adhesion, herein we describe the generation of high-density peptide arrays and test the growth of cells on these multifunctionalized surfaces. The peptide library used consists of over 11,000 different sequences, either random or derived from existing proteins. By applying this screen to SW620 mCherry colorectal cancer cells, we select for peptides with both maximum cell adhesion and maximum cell repulsion. All of these extreme properties are based on unique combinations of amino acids. Here, we identify peptides with maximum cell repulsion on secreted frizzled- and Dickkopf-related proteins. Peptides with strong cell repulsion are found at the poles of the TNF-alpha homotrimer. The formation of cellular patterns on alternating highly repulsive and adhesive peptides are examined. Our screen allows the identification of peptides suitable for biomedical and tissue engineering applications.

Phage Display Panning on Silica: Optimization of Elution Conditions for Selection of Strong Binders.

Phage display panning is a powerful tool to select strong peptide binders to a given target, and when applied to inorganic materials (e.g., silica) as a target, it provides information on binding events and molecular recognition at the peptide-mineral interface. The panning process has limitations with the phage chemical elution being affected by bias toward positively charged binders, resulting in the potential loss of information on binder diversity; the presence of fast growing phages with an intrinsic growth advantage; and the presence of false positives from target unrelated peptides. To overcome some of these limitations, we developed a panning approach based on the sequential use of different eluents (Gly-HCl, pH-2.2; MgCl2, pH-6.1; and TEA, pH-11.0), or pH conditions (Gly-HCl 2.2 < pH < 11.0) that allows the identification of a diverse and comprehensive pool of strong binders. We have assessed and tested the authenticity of the identified silica binders via a complementary experimental (in vivo phage recovery rates and TEM imaging) and bioinformatics approach. We provide experimental evidence of the nonspecificity of the Gly-HCl eluent as typically used. Using a fluorimetric assay, we investigate in vitro binding of two peptides that differ by pI-S4 (HYIDFRW, pI 7.80) and S5 (YSLKQYQ, pI 9.44)─modified at the C terminal with an amide group to simulate net charges in the phage display system, confirming the vital role of electrostatic interactions as driving binding forces in the phage panning process. The presented optimized phage panning approach provides an opportunity to match known surface interactions at play with suitable elution conditions; to select only sequences relevant to a particular interfacial system. The approach has the potential to open up avenues to design interfacial systems to advance our understanding of peptide-assisted mineral growth, among other possibilities.

Activation of Polycystin-1 Signaling by Binding of Stalk-derived Peptide Agonists.

Polycystin-1 (PC1) is the membrane protein product of the PKD1 gene whose mutation is responsible for 85% of the cases of autosomal dominant polycystic kidney disease (ADPKD). ADPKD is primarily characterized by the formation of renal cysts and potential kidney failure. PC1 is an atypical G protein-coupled receptor (GPCR) consisting of 11 transmembrane helices and an autocatalytic GAIN domain that cleaves PC1 into extracellular N-terminal (NTF) and membrane-embedded C-terminal (CTF) fragments. Recently, signaling activation of the PC1 CTF was shown to be regulated by a stalk tethered agonist (TA), a distinct mechanism observed in the adhesion GPCR family. A novel allosteric activation pathway was elucidated for the PC1 CTF through a combination of Gaussian accelerated molecular dynamics (GaMD), mutagenesis and cellular signaling experiments. Here, we show that synthetic, soluble peptides with 7 to 21 residues derived from the stalk TA, in particular, peptides including the first 9 residues (p9), 17 residues (p17) and 21 residues (p21) exhibited the ability to re-activate signaling by a stalkless PC1 CTF mutant in cellular assays. To reveal molecular mechanisms of stalk peptide-mediated signaling activation, we have applied a novel Peptide GaMD (Pep-GaMD) algorithm to elucidate binding conformations of selected stalk peptide agonists p9, p17 and p21 to the stalkless PC1 CTF. The simulations revealed multiple specific binding regions of the stalk peptide agonists to the PC1 protein including an "intermediate" bound yet inactive state. Our Pep-GaMD simulation findings were consistent with the cellular assay experimental data. Binding of peptide agonists to the TOP domain of PC1 induced close TOP-putative pore loop interactions, a characteristic feature of the PC1 CTF signaling activation mechanism. Using sequence covariation analysis of PC1 homologs, we further showed that the peptide binding regions were consistent with covarying residue pairs identified between the TOP domain and the stalk TA. Therefore, structural dynamic insights into the mechanisms of PC1 activation by stalk-derived peptide agonists have enabled an in-depth understanding of PC1 signaling. They will form a foundation for development of PC1 as a therapeutic target for the treatment of ADPKD.

Structure-Guided Antiviral Peptides Identification Targeting the HIV-1 Integrase.

HIV-1 integrase (IN), a major protein in the HIV life cycle responsible for integrating viral cDNA into the host DNA, represents a promising drug target. Small peptides have emerged as antiviral therapeutics for HIV because of their facile synthesis, highly selective nature, and fewer side effects. However, selecting the best candidates from a vast pool of peptides is a daunting task. In this study, multistep virtual screening was employed to identify potential peptides from a list of 280 HIV inhibitory peptides. Initially, 80 peptides were selected based on their minimum inhibitory concentrations (MIC). Then, molecular docking was performed to evaluate their binding scores compared to HIP000 and HIP00N which are experimentally validated HIV-1 integrase binding peptides that were used as a positive and negative control, respectively. The top-scoring docked complexes, namely, IN-HIP1113, IN-HIP1140, IN-HIP1142, IN-HIP678, IN-HIP776, and IN-HIP777, were subjected to initial 500 ns molecular dynamics (MD) simulations. Subsequently, HIP776, HIP777, and HIP1142 were selected for an in-depth mechanistic study of peptide interactions, with multiple simulations conducted for each complex spanning one microsecond. Independent simulations of the peptides, along with comparisons to the bound state, were performed to elucidate the conformational dynamics of the peptides. These peptides exhibit strong interactions with specific residues, as revealed by snapshot interaction analysis. Notably, LYS159, LYS156, VAL150, and GLU69 residues are prominently involved in these interactions. Additionally, residue-based binding free energy (BFE) calculations highlight the significance of HIS67, GLN148, GLN146, and SER147 residues within the binding pocket. Furthermore, the structure-activity relationship (SAR) analysis demonstrated that aromatic amino acids and the overall volume of peptides are the two major contributors to the docking scores. The best peptides will be validated experimentally by incorporating SAR properties, aiming to develop them as therapeutic agents and structural models for future peptide-based HIV-1 drug design, addressing the urgent need for effective HIV treatments.

The spatial separation of basic amino acids is similar in RHAMM and hyaluronan binding peptide P15-1 despite different sequences and conformations.

Peptides that increase pro-reparative responses to injury and disease by modulating the functional organization of hyaluronan (HA) with its cell surface binding proteins (e.g., soluble receptor for hyaluronan-mediated motility [RHAMM] and integral membrane CD44) have potential therapeutic value. The binding of RHAMM to HA is an attractive target, since RHAMM is normally absent or expressed at low levels in homeostatic conditions, but its expression is significantly elevated in the extracellular matrix during tissue stress, response-to-injury, and in cancers and inflammation-based diseases. The HA-binding site in RHAMM contains two closely spaced sequences of clustered basic amino acids, in an alpha-helical conformation. In the present communication, we test whether an alpha-helical conformation is required for effective peptide binding to HA, and competitive disruption of HA-RHAMM interaction. The HA-binding RHAMM-competitive peptide P15-1, identified using the unbiased approach of phage display, was examined using circular dichroism spectroscopy and the conformation-predictive AI-based AlphaFold2 algorithm. Unlike the HA-binding site in RHAMM, peptide P15-1 was found to adopt irregular conformations in solution rather than alpha helices. Instead, our structural analysis suggests that the primary determinant of peptide-HA binding is associated with a specific clustering and spacing pattern of basic amino acids, allowing favorable electrostatic interaction with carboxylate groups on HA.