Peptide Binding Research Articles

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.

Hemoglobin Hydrolyzate Promotes Iron Absorption in the Small Intestine through Iron Binding Peptides.

Hemoglobin is an excellent source of iron supplements, and its hydrolyzate spontaneously binds iron during digestion and promotes iron absorption in vivo. However, the underlying mechanisms of what peptides bind and how they bind iron ions remain unclear. This study prepared the porcine hemoglobin hydrolyzate through enzymatic hydrolysis and acid treatment and investigated the mechanisms of hemoglobin hydrolyzate on iron absorption through the determination of iron levels in dietary intervention mice, iron binding site analyses, peptide digestion analyses, molecular simulation docking, and INT407 cell validation. The results showed that ingestion of the hemoglobin hydrolyzate diets increased iron levels in the blood of mice, accompanied by the upregulation of duodenal iron circulation-related genes such as ferritin, PCBP1, and HP. Carboxyl, imidazole groups, and aromatic amino acid residues were iron binding sites of hemoglobin hydrolyzate during digestion. VDEVGGEA and VDEVGGE were found to involve the spontaneous and efficient binding of hemoglobin hydrolyzate to iron ions in the intestinal cavity. In particular, the DEVGGE peptide was the typical sequence for hemoglobin hydrolytic peptides to exert iron binding activity.

Development of a nanoparticle-based tendon-targeting drug delivery system to pharmacologically modulate tendon healing.

Satisfactory healing following acute tendon injury is marred by fibrosis. Despite the high frequency of tendon injuries and poor outcomes, there are no pharmacological therapies in use to enhance the healing process. Moreover, systemic treatments demonstrate poor tendon homing, limiting the beneficial effects of potential tendon therapeutics. To address this unmet need, we leveraged our existing tendon healing spatial transcriptomics dataset and identified an area enriched for expression of Acp5 (TRAP) and subsequently demonstrated robust TRAP activity in the healing tendon. This unexpected finding allowed us to refine and apply our existing TRAP binding peptide (TBP) functionalized nanoparticle (NP) drug delivery system (DDS) to facilitate improved delivery of systemic treatments to the healing tendon. To demonstrate the translational potential of this DDS, we delivered niclosamide (NEN), an S100a4 inhibitor. While systemic delivery of free NEN did not alter healing, TBP-NPNEN enhanced both functional and mechanical recovery, demonstrating the translational potential of this approach to enhance the tendon healing process.

A non-B DNA binding peptidomimetic channel alters cellular functions

DNA binding transcription factors possess the ability to interact with lipid membranes to construct ion-permeable pathways. Herein, we present a thiazole-based DNA binding peptide mimic TBP2, which forms transmembrane ion channels, impacting cellular ion concentration and consequently stabilizing G-quadruplex DNA structures. TBP2 self-assembles into nanostructures, e.g., vesicles and nanofibers and facilitates the transportation of Na+ and K+ across lipid membranes with high conductance (~0.6 nS). Moreover, TBP2 exhibits increased fluorescence when incorporated into the membrane or in cellular nuclei. Monomeric TBP2 can enter the lipid membrane and localize to the nuclei of cancer cells. The coordinated process of time-dependent membrane or nuclear localization of TBP2, combined with elevated intracellular cation levels and direct G-quadruplex (G4) interaction, synergistically promotes formation and stability of G4 structures, triggering cancer cell death. This study introduces a platform to mimic and control intricate biological functions, leading to the discovery of innovative therapeutic approaches.

Revealing molecular determinants governing mambalgin-3 pharmacology at acid-sensing ion channel 1 variants

Acid-sensing ion channels (ASICs) are trimeric proton-gated cation channels that play a role in neurotransmission and pain sensation. The snake venom-derived peptides, mambalgins, exhibit potent analgesic effects in rodents by inhibiting central ASIC1a and peripheral ASIC1b. Despite their distinct species- and subtype-dependent pharmacology, previous structure-function studies have focussed on the mambalgin interaction with ASIC1a. Currently, the specific channel residues responsible for this pharmacological profile, and the mambalgin pharmacophore at ASIC1b remain unknown. Here we identify non-conserved residues at the ASIC1 subunit interface that drive differences in the mambalgin pharmacology from rat ASIC1a to ASIC1b, some of which likely do not make peptide binding interactions. Additionally, an amino acid variation below the core binding site explains potency differences between rat and human ASIC1. Two regions within the palm domain, which contribute to subtype-dependent effects for mambalgins, play key roles in ASIC gating, consistent with subtype-specific differences in the peptides mechanism. Lastly, there is a shared primary mambalgin pharmacophore for ASIC1a and ASIC1b activity, with certain peripheral peptide residues showing variant-specific significance for potency. Through our broad mutagenesis studies across various species and subtype variants, we gain a more comprehensive understanding of the pharmacophore and the intricate molecular interactions that underlie ligand specificity. These insights pave the way for the development of more potent and targeted peptide analogues required to advance our understating of human ASIC1 function and its role in disease.

Mts1 (S100A4) and Its Peptide Demonstrate Cytotoxic Activity in Complex with Tag7 (PGLYRP1) Peptide.

Receptors of cytokines are major regulators of the immune response. In this work, we have discovered two new ligands that can activate the TNFR1 (tumor necrosis factor receptor 1) receptor. Earlier, we found that the peptide of the Tag (PGLYRP1) protein designated 17.1 can interact with the TNFR1 receptor. Here, we have found that the Mts1 (S100A4) protein interacts with this peptide with a high affinity (Kd = 1.28 × 10-8 M), and that this complex is cytotoxic to cancer cells that have the TNFR1 receptor on their surface. This complex induces both apoptosis and necroptosis in cancer cells with the involvement of mitochondria and lysosomes in cell death signal transduction. Moreover, we have succeeded in locating the Mts1 fragment that is responsible for protein-peptide interaction, which highly specifically interacts with the Tag7 protein (Kd = 2.96 nM). The isolated Mts1 peptide M7 also forms a complex with 17.1, and this peptide-peptide complex also induces the TNFR1 receptor-dependent cell death. Molecular docking and molecular dynamics experiments show the amino acids involved in peptide binding and that may be used for peptidomimetics' development. Thus, two new cytotoxic complexes were created that were able to induce the death of tumor cells via the TNFR1 receptor. These results may be used in therapy for both cancer and autoimmune diseases.

Competitive Binding of Viral Nuclear Localization Signal Peptide and Inhibitor Ligands to Importin-α Nuclear Transport Protein.

Venezuelan equine encephalitis virus (VEEV) is a highly virulent pathogen whose nuclear localization signal (NLS) sequence from capsid protein binds to the host importin-α transport protein and blocks nuclear import. We studied the molecular mechanisms by which two small ligands, termed I1 and I2, interfere with the binding of VEEV's NLS peptide to importin-α protein. To this end, we performed all-atom replica exchange molecular dynamics simulations probing the competitive binding of the VEEV coreNLS peptide and I1 or I2 ligand to the importin-α major NLS binding site. As a reference, we used our previous simulations, which examined noncompetitive binding of the coreNLS peptide or the inhibitors to importin-α. We found that both inhibitors completely abrogate the native binding of the coreNLS peptide, forcing it to adopt a manifold of nonnative loosely bound poses within the importin-α major NLS binding site. Both inhibitors primarily destabilize the native coreNLS binding by masking its amino acids rather than competing with it for binding to importin-α. Because I2, in contrast to I1, binds off-site localizing on the edge of the major NLS binding site, it inhibits fewer coreNLS native binding interactions than I1. Structural analysis is supported by computations of the free energies of the coreNLS peptide binding to importin-α with or without competition from the inhibitors. Specifically, both inhibitors reduce the free energy gain from coreNLS binding, with I1 causing significantly larger loss than I2. To test our simulations, we performed AlphaScreen experiments measuring IC50 values for both inhibitors. Consistent with in silico results, the IC50 value for I1 was found to be lower than that for I2. We hypothesize that the inhibitory action of I1 and I2 ligands might be specific to the NLS from VEEV's capsid protein.

Modeling the Binding of Anticancer Peptides and Mcl-1.

Mcl-1 (myeloid cell leukemia 1), a member of the Bcl-2 family, is upregulated in various types of cancer. Peptides representing the BH3 (Bcl-2 homology 3) region of pro-apoptotic proteins have been demonstrated to bind the hydrophobic groove of anti-apoptotic Mcl-1, and this interaction is responsible for regulating apoptosis. Structural studies have shown that, while there is high overall structural conservation among the anti-apoptotic Bcl-2 (B-cell lymphoma 2) proteins, differences in the surface groove of these proteins facilitates binding specificity. This binding specificity is crucial for the mechanism of action of the Bcl-2 family in regulating apoptosis. Bim-based peptides bind specifically to the hydrophobic groove of Mcl-1, emphasizing the importance of these interactions in the regulation of cell death. Molecular docking was performed with BH3-like peptides derived from Bim to identify high affinity peptides that bind to Mcl-1 and to understand the molecular mechanism of their interactions. The interactions of three identified peptides, E2gY, E2gI, and XXA1_F3dI, were further evaluated using 250 ns molecular dynamics simulations. Conserved hydrophobic residues of the peptides play an important role in their binding and the structural stability of the complexes. Understanding the molecular basis of interaction of these peptides will assist in the development of more effective Mcl-1 specific inhibitors.

All-atom modeling of methacrylate-based multi-modal chromatography resins for Langmuir constant prediction of peptides

In downstream processing, the intricate nature of the interactions between biomolecules and adsorbent materials presents a significant challenge in the prediction of their binding and elution behaviors. This complexity is further heightened in multi-modal chromatography (MMC), which employs two distinct binding mechanisms. To gain a deeper understanding of the involved interactions, simulating the adsorption of biomolecules on resin surfaces is a focal point of ongoing research. However, previous studies often simplified the adsorbent surface, modeling it as a flat or slightly curved plane without including a realistic backbone structure. Here, we introduce and validate two novel workflows aimed at predicting peptide binding behaviors in MMC, specifically targeting methacrylate-based resins. Our first achievement was the development of an all-atom model of a commercial MMC resin surface, incorporating its polymethacrylic backbone. Furthermore, we established and tested a workflow for rapid calculations of binding free energies (ΔG) with 10 linear peptides as target molecules. These ΔG calculations were effectively used to predict Langmuir constants, achieving a high coefficient of determination (R²) of 0.96. In subsequent benchmarking tests, our model outperformed established, simpler resin surface models in terms of predictive capabilities.

Fluorescein-Based SynNotch Adaptors for Regulating Gene Expression Responses to Diverse Extracellular Cues.

We introduce an adaptor-based strategy for regulating fluorescein-binding synthetic Notch (SynNotch) receptors using ligands based on conjugates of fluorescein isomers and analogs. To develop a versatile system, we evaluated the surface expression and activities of multiple constructs containing distinct extracellular fluorescein-binding domains. Using an optimized receptor, we devised ways to regulate signaling via fluorescein-based chemical transformations, including an approach based on a bio-orthogonal chemical ligation and a spatially controllable strategy via the photo-patterned uncaging of an o -nitrobenzyl-caged fluorescein conjugate. We further demonstrate that fluorescein-conjugated extracellular matrix (ECM)-binding peptides can regulate SynNotch activity depending on the folding state of collagen-based ECM networks. Treatment with these conjugates enabled cells to distinguish between folded versus denatured collagen proteins and enact dose-dependent gene expression responses depending on the nature of the signaling adaptors presented. To demonstrate the utility of these tools, we applied them to control the myogenic conversion of fibroblasts into myocytes with spatial and temporal precision and in response to denatured collagen-I, a biomarker of multiple pathological states. Overall, we introduce an optimized fluorescein-binding SynNotch as a versatile tool for regulating transcriptional responses to extracellular ligands based on the widely used and clinically-approved fluorescein dye.

DP-site: A dual deep learning-based method for protein-peptide interaction site prediction

BackgroundProtein-peptide interaction prediction is an important topic for several applications including various biological processes, understanding drug discovery, protein function abnormal cellular behaviors, and treating diseases. Over the years, studies have shown that experimental methods have improved the identification of this bio-molecular interaction. However, predicting protein-peptide interactions using these methods is laborious, time-consuming, dependent on third-party tools, and costly. MethodTo address these previous drawbacks, this study introduces a computational framework called DP-Site. The proposed framework concentrates on using a compound of a dual pipeline along with a combination predictor. A deep convolutional neural network for feature extraction and classification is embedded in pipeline 1. In addition, pipeline 2 includes a deep long-short-term memory-based and a random forest classifier for feature extraction and classification. In this investigation, the evolutionary, structure-based, sequence-based, and physicochemical information of proteins is utilized for identifying protein-peptide interaction at the residue level. ResultsThe proposed method is evaluated on both the ten-fold cross-validation and independent test sets. The robust and consistent results between cross-validation and independent test sets confirm the ability of the proposed method to predict peptide binding residues in proteins. Moreover, experimental findings demonstrate that DP-Site has significantly outperformed other state-of-the-art sequence-based and structure-based methods. The proposed method achieves a remarkable balance between a specificity of 0.799 and a sensitivity of 0.770, along with the best f-measure of 0.661 and the highest precision of 0.580 using an independent test set. ConclusionsThe outcome of various experiments confirms the proficiency of the proposed method and outperforms state-of-the-art sequence-based and structure-based methods in terms of the mentioned criteria. DP-Site can be accessed at https://github.com/shafiee 95/shima.shafiee.DP-Site.

A universal method for surface-based binding assays by preparing immobilized β2-adrenergic receptor stationary phase using solid binding peptide as a fusion tag

Protein functionalized surface has the potential to develop new assays for determining the drug-like properties of potential compounds and discovering specific partners of G protein-coupled receptors (GPCRs). However, a universal method for purifying and immobilizing functional GPCRs has remained elusive. To this end, we developed a general and rapid way to purify and immobilize β2-adrenergic receptor (β2AR) by silicon-specific peptide. We screened CotB1p as a tag from six silica-binding peptides (minTBP-1, CotB1p, SB7, Car9, and Si4–1) by examining their affinity to macroporous silica gel. We investigated the adsorption and desorption of CotB1p-tagged β2-adrenoceptor (β2AR-CotB1p) under diverse conditions to propose a protocol for receptor purification and immobilization. Under optimized conditions, β2AR immobilization were achieved by directly immersing cell lysates harboring the receptor with silica gel, and the elution of the receptor without demonstratable contaminants was realized by including l-arginine/L-lysine in the elutes. This allows purification of the receptor from Escherichia coli (E.coli) lysates with a purity of 95 %. The immobilized receptor was utilized as a stationary phase to reveal the tag impact on ligand-binding outputs by comparing the CotB1p-strategy with a typical covalent method. The KAs of salbutamol, chlorprenaline, tulobuterol, and terbutaline on β2AR-CotB1p column were 1.26 × 106, 6.59 × 106, 7.90 × 106, and 8.97 × 105 M−1 respectively, which were two orders of magnitude higher than those on the Halo-β2AR column. The whole immobilization was accomplished within 30 min without the requirement of any special treatment, resulting in enhanced accuracy for determining receptor-ligand binding parameters. Taken together, CotB1p-mediated strategy is simple, rapid, and universal for purification or immobilization of unstable biomolecules like GPCRs for analytical and biological applications.

Discovery of a new class of cell-penetrating peptides by novel phage display platform

The primary hurdles for small interference RNA (siRNA) in clinical use are targeted and cytosolic delivery. To overcome both challenges, we have established a novel platform based on phage display, called NNJA. In this approach, a lysosomal cathepsin substrate is engineered within the flexible loops of PIII, that is displaying a unique random sequence at its N-terminus. NNJA library selection targeting cell-expressed targets should yield specific peptides localized in the cytoplasm. That is because phage internalization and subsequent localization to lysosome, upon peptide binding to the cell expressed target, will result in cleavage of PIII, rendering phage non-infective. Such phage will be eliminated from the selected pool and only peptide-phage that escapes lysosomes will advance to the next round. Proof of concept studies with the NNJA library demonstrated cytosolic localization of selected peptide-phage and peptide-siRNA, confirmed through confocal microscopy. More importantly, conjugation of siHPRT to monomeric or multimeric NNJA peptides resulted in significant reduction in HPRT mRNA in various cell types without significant cytotoxicity. Sequence similarity and clustering analysis from NGS dataset provide insights into sequence composition facilitating cell penetration. NNJA platform offers a highly efficient peptide discovery engine for targeted delivery of oligonucleotides to cytosol.

Phosphorylation-responsive system for enzyme activity detection based on peptide-cucurbit[8]uril interactions

Cucurbit [8]uril (CB[8]) exhibits high binding affinity to specific peptide sequences, allowing the peptide-CB[8] system to operate effectively in diverse biological applications. However, it is imperative to identify effective regulation factors for the binding of peptides with CB[8], making it a more promising and responsive host-guest system. In this study, we discovered that phosphorylation at the side chain of tyrosine residue can completely disrupt the binding of a tripeptide motif (YLA or MYA) with CB[8]. This discovery introduces a novel enzymatic strategy for regulating the peptide-CB[8] host-guest interaction. Furthermore, it has been applied to detect the activity of phosphatases by supramolecular tandem assays.

Peptide binding classification on quantum computers

Peptide binding classification on quantum computers

Understanding Aβ Peptide Binding to Lipid Membranes: A Biophysical Perspective.

Aβ peptides are known to bind neural plasma membranes in a process leading to the deposit of Aβ-enriched plaques. These extracellular structures are characteristic of Alzheimer's disease, the major cause of late-age dementia. The mechanisms of Aβ plaque formation and deposition are far from being understood. A vast number of studies in the literature describe the efforts to analyze those mechanisms using a variety of tools. The present review focuses on biophysical studies mostly carried out with model membranes or with computational tools. This review starts by describing basic physical aspects of lipid phases and commonly used model membranes (monolayers and bilayers). This is followed by a discussion of the biophysical techniques applied to these systems, mainly but not exclusively Langmuir monolayers, isothermal calorimetry, density-gradient ultracentrifugation, and molecular dynamics. The Methodological Section is followed by the core of the review, which includes a summary of important results obtained with each technique. The last section is devoted to an overall reflection and an effort to understand Aβ-bilayer binding. Concepts such as Aβ peptide membrane binding, adsorption, and insertion are defined and differentiated. The roles of membrane lipid order, nanodomain formation, and electrostatic forces in Aβ-membrane interaction are separately identified and discussed.

A Top-Down Design Approach for Generating a Peptide PROTAC Drug Targeting Androgen Receptor for Androgenetic Alopecia Therapy.

While large-scale artificial intelligence (AI) models for protein structure prediction and design are advancing rapidly, the translation of deep learning models for practical macromolecular drug development remains limited. This investigation aims to bridge this gap by combining cutting-edge methodologies to create a novel peptide-based PROTAC drug development paradigm. Using ProteinMPNN and RFdiffusion, we identified binding peptides for androgen receptor (AR) and Von Hippel-Lindau (VHL), followed by computational modeling with Alphafold2-multimer and ZDOCK to predict spatial interrelationships. Experimental validation confirmed the designed peptide's binding ability to AR and VHL. Transdermal microneedle patching technology was seamlessly integrated for the peptide PROTAC drug delivery in androgenic alopecia treatment. In summary, our approach provides a generic method for generating peptide PROTACs and offers a practical application for designing potential therapeutic drugs for androgenetic alopecia. This showcases the potential of interdisciplinary approaches in advancing drug development and personalized medicine.

Molecular docking and molecular dynamics simulation of wheat gluten-derived antioxidant peptides acting through the Keap1-Nrf2 pathway.

In our previous study, we successfully identified five peptides from wheat gluten: Ala-Pro-Ser-Tyr (APSY), Leu-Tyr (LY), Pro-Tyr (PY), Arg-Gly-Gly-Tyr (RGGY) and Tyr-Gln (YQ). Molecular docking and molecular dynamics simulation methods were employed to investigate the interaction between these antioxidant peptides and the Kelch-like ECH-associated protein 1 (Keap1 protein), revealing the molecular mechanism of their non-competitive binding. In addition, the total antioxidant capacity of the five peptides was determined using the 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) method. The affinities of APSY, LY, PY, RGGY and YQ were -8.9, -8.3, -8.5, -9.1 and - 7.9 kcal mol-1, respectively. The five peptides effectively bound to Keap1 protein through hydrogen, π-σ, π-alkyl and alkyl interactions. Significant roles were observed for the P1 pocket residue ARG-415 and the P3 pocket residue ALA-556 in the interactions of the Keap1-peptide complexes. Molecular dynamics simulations further elucidated the dynamic process of peptide binding to the Keap1 protein. All five peptides formed stable complexes with Keap1 protein, with van der Waals forces playing crucial roles in these complex systems, indicative of the peptides' strong binding ability to Keap1 protein. The van der Waals forces were -178.74, -123.11, -134.36, -132.59, and -121.44 kJ mol-1 for the Keap1-APSY, Keap1-LY, Keap1-PY, Keap1-RGGY and Keap1-YQ complexes, respectively. These peptides exhibited excellent antioxidant effects. Among them, the YQ peptide exhibited the highest total antioxidant capacity, with an activity value of 1.18 ± 0.06 mmol Trolox equivalent (TE) L-1 at a concentration of 0.10 mg mL-1. The RGGY, PY, LY and APSY peptides followed in descending order, with activity values of 0.91 ± 0.05, 0.72 ± 0.06, 0.62 ± 0.04 and 0.60 ± 0.05 mmol TE L-1, respectively. These results unveiled the molecular mechanism by which the five antioxidant peptides act on active pockets through the Keap1-Nrf2 signaling pathway, providing a theoretical basis for the development of antioxidants. © 2024 Society of Chemical Industry.

Covalent Flexible Peptide Docking in Drug Discovery: Current Challenges and Potential Interventions

Recently, there has been a significant increase in the search for new covalent inhibitors through drug discovery platforms, which has led to the development and implementation of new computational tools, including covalent docking methods to predict the binding mode and affinity of covalent ligands. Since the discovery of insulin nearly a century ago, more than 80 peptide medications have hit the market to treat a wide range of diseases, including diabetes, cancer, osteoporosis, multiple sclerosis, HIV infection, and chronic pain. Electrophilic peptides that form covalent bonds with target proteins have great potential for binding targets that have been previously considered undruggable. Despite the recent advancements in computational performance and docking algorithms, covalent docking still poses a number of challenges. Peptides covalent docking presents additional challenges, the main ones being the choice of the optimal peptide sequence, incorporation of electrophilic warheads, the inherent flexibility of peptide structures, and the fact that, unlike small molecules, peptides do fold. In this review, we present a brief overview of the current state of peptide therapeutics in drug discovery, covalent docking in general, covalent peptide binders, and-with particular emphasis-the difficulties that covalent peptide-protein docking is currently facing and some potential solutions.

Uncommon P1 Anchor-featured Viral T Cell Epitope Preference within HLA-A*2601 and HLA-A*0101 Individuals.

The individual HLA-related susceptibility to emerging viral diseases such as COVID-19 underscores the importance of understanding how HLA polymorphism influences peptide presentation and T cell recognition. Similar to HLA-A*0101, which is one of the earliest identified HLA alleles among the human population, HLA-A*2601 possesses a similar characteristic for the binding peptide and acts as a prevalent allomorph in HLA-I. In this study, we found that, compared with HLA-A*0101, HLA-A*2601 individuals exhibit distinctive features for the T cell responses to SARS-CoV-2 and influenza virus after infection and/or vaccination. The heterogeneous T cell responses can be attributed to the distinct preference of HLA-A*2601 and HLA-A*0101 to T cell epitope motifs with negative-charged residues at the P1 and P3 positions, respectively. Furthermore, we determined the crystal structures of the HLA-A*2601 complexed to four peptides derived from SARS-CoV-2 and human papillomavirus, with one structure of HLA-A*0101 for comparison. The shallow pocket C of HLA-A*2601 results in the promiscuous presentation of peptides with "switchable" bulged conformations because of the secondary anchor in the median portion. Notably, the hydrogen bond network formed between the negative-charged P1 anchors and the HLA-A*2601-specific residues lead to a "closed" conformation and solid placement for the P1 secondary anchor accommodation in pocket A. This insight sheds light on the intricate relationship between HLA I allelic allomorphs, peptide binding, and the immune response and provides valuable implications for understanding disease susceptibility and potential vaccine design.