Cross‐reaction mediated by distinct key amino acid combinations in the complementary‐determining region (CDR) of a monoclonal antibody

In immunology, cross‐reaction between antigens and antibodies are commonly observed. Prior research has shown that various monoclonal antibodies (mAbs) can recognize a broad spectrum of epitopes related to influenza viruses. However, existing theories on cross‐reactions fall short in explaining the phenomena observed. This study explored the interaction characteristics of H1‐74 mAb with three peptides: two natural peptides, LVLWGIHHP and LPFQNI, derived from the hemagglutinin (HA) antigen of the H1N1 influenza virus, and one synthetic peptide, WPFQNY. Our findings indicate that the complementarity‐determining region (CDR) of H1‐74 mAb comprised five antigen‐binding sites, containing eight key amino acid residues from the light chain variable region and 16 from the heavy chain variable region. These critical residues formed distinct hydrophobic or hydrophilic clusters and functional groups within the binding sites, facilitating interaction with antigen epitopes through hydrogen bonding, salt bridge formation, and π–π stacking. The study revealed that the formation of the antibody molecule led to the creation of binding groups and small units in the CDR, allowing the antibody to attach to a variety of antigen epitopes through diverse combinations of these small units and functional groups. This unique ability of the antibody to bind with antigen epitopes provides a new molecular basis for explaining the phenomenon of antibody cross‐reaction.


| INTRODUCTION
Antibodies are the culmination of the immune system's response to foreign antigens.They interact with antigens in a highly specific manner, recognizing antigenic determinants or epitopes, which include linear and conformational epitopes.These epitopes correspond to complementary determinants, known as paratopes, on the antibody. 1 1936, Karl Landsteiner first observed that antigens and antibodies could cross-react, in addition to their specific reactions. 2is phenomenon suggests that, beyond their specific antigenic epitopes, many antigens share common antigen epitopes, termed coantigens.Typically, cross-reaction occurs due to linear epitopes with identical sequences or conformational epitopes with similar structure. 3For nearly a century, numerous pathogens and their products have been reported to cross-react with human tissues, potentially inducing autoimmune diseases.This cross-reactivity involves common epitopes between autoantigens in autoimmune diseases and natural microbial proteins; these are either linear epitopes with the same sequence or conformational epitopes with the same or similar structure. 4These include the PPPGRRP peptide of Epstein-Barr virus nuclear antigen-1 and the spliceosome SmB/B′ peptide in systemic lupus erythematosus patients, 5 along with epitopes found in colon tissue and Escherichia coli O14. 6Further, the overlapping sequence between the M5 protein (85-103) region of Streptococcus and cardiac myosin results in the cross-reactivity between streptococcus M5 protein and cardiac histonein. 7wever, cross-reactivity can also occur in the absence of confirmed co-antigens in certain autoimmune disorders.For example, antibodies produced to human T-lympho virus type 1 (HTLV-1) infection can interact with hnRNPA1, leading to myelopathy/tropical spastic paraplegia.Levin's use of the mimotope multipin peptide system identified the dominant epitope KHFRETEV in HTLV-1, which does not correspond to any similar sequence or conformational epitopes on hnRNPA1. 8Similarly, monoclonal antibodies (mAbs) of H1N1 influenza virus hemagglutinin (HA) have been found to interact with islet cell prohibition proteins without any homologous sequence or conformational epitopes with similar structures between HA and prohibition proteins. 9In most cases, there is no homologous sequence or conformational epitopes with similar structure between autoantigens and microbial antigens, and this phenomenon remains unexplained.Consequently, direct evidence linking pathogenic microbial infection and autoimmune disease is limited, impacting early disease diagnosis and treatment.
A previous study focusing on the localization of anti-2009 H1N1 influenza virus mAb binding to a target antigen, 10  The essential residues from antigens forming epitopes are termed "immunodominant groups."The key amino acids in the variable region of an antibody that interact with these immunodominant groups are referred to as "key amino acid residues," for distinction.

Detailed descriptions of the experiments are provided in Supporting
Information S1: Sections S1 and S2.

| Distribution of polypeptides LP-9 and LI-6 on the HA crystal structure
The HA crystal structure of the influenza A (H1N1) virus was obtained from the PDB database.The 3LZG protein sequence was referenced to locate the distribution regions of LP-9 and LI-6 on the HA crystal using PyMOL software.

| Cloning, sequencing, and molecular modeling of H1-74 mAb's light and heavy chain variable regions
Following established procedures, messenger RNA was extracted from H1-74 mAb hybridoma cells 12 and converted into complementary DNA for gene cloning.The variable regions of the light and heavy chains of H1-74 mAb were cloned, sequenced, and molecularly modeled (Supporting Information S1: Sections S2.1 and S2.2).

| Identification (LXXXI) of amino acid substitutions for LVLWGIHHP skeleton in 6-peptide LPFQNI that maintain H1-74 mAb binding through molecular simulation
Using MOE software, we analyzed the dominant conformational structures of the LI-6 peptide chain.Antibody-antigen docking provided a reliable model of their interactions.Docking was performed using Amber10, and the most effective antigenantibody binding mode was selected for analyzing amino acid mutations in 6-peptide LPFQNI.Virtual mutation methods were employed to identify mutants in LI-6 that affected binding activity with H1-74 mAb (Supporting Information S1: Sections S2.3 and S2.4).

| Analysis of the binding activity of mutant polypeptides
Enzyme-linked immunosorbent assay (ELISA) plates were coated with H1N1 influenza virus HA antigen at a concentration of 2 μg/mL.
Peptides LP-9, LI-6, the enhanced mutant peptide WY-6, and the attenuated mutant peptide LPGQGI were incubated with H1-74 mAb at 37°C for 1 h.The mixture was added to the HA-coated ELISA plates.The assay was conducted using standard ELISA procedures (100 μL per well, 37°C for 1 h).The mixture was washed and then sheep anti-mouse enzyme-labeled antibody was added to it.The other steps were the same as in conventional ELISA.Using TMB color rendering, the OD 450 nm value of each well was determined.The inhibition rate was calculated according to the OD 450 nm value of each well 12,13 : inhibition rate = (untreated control group − experimental group)/untreated control group.An inhibition rate ≤0.4 indicated nonrecognition of the epitope by the antibody.Rates 0.4 and 0.8 suggested partial recognition and ≥0.8 indicated full recognition.
2.6 | Characterization of H1-74 binding to LP-9, LI-6, and WY-6 Three-dimensional structural models of LP-9, LI-6, and WY-6 were constructed using UCSF Chimera software.Initial molecular dynamics simulations were performed to determine the rational structural conformations of three polypeptides.Flexible molecular docking of H1-74 mAb with these peptides was then executed using Rosetta software.Both proteins and peptides were set as flexible for 100 docking conformations.The conformation with the lowest docking energy for each peptide (LP-9, LI-6, and WY-6)-H1-74 pairing was selected for further molecular dynamics simulation using Amber16 (Supporting Information S1: Section S2.5).

| Preparation and characterization of monoclonal and polyclonal antibodies using LP-9, LI-6, and WY-6 as immunogens
Consistent with prior research, peptides were conjugated to bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH) carrier proteins 14,15 using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) activators for immunization.We used EDC to activate the carboxyl terminals of three polypeptides, coupled with the carrier proteins BSA and KLH. 16The cross-linking of the polypeptides to the carriers was outsourced to Shanghai Qiangyao Biological Company.
The preparation and characterization of both monoclonal and polyclonal antibodies are described in Supporting Information S1: Sections S2.6-S2.10.
We extracted the amino acid sequences of the variable regions of H1-74 mAb's light and heavy chains.Analysis using abYsis software (http://www.abysis.org/abysis/)indicated distinct structural features in the skeletal (FR) and variable (CDR) regions of the light and heavy chains of H1-74 (Figure 1B).Sequence analysis against the RCSB PBD database, followed by homology modeling for H1-74 mAb using Modeller software, selected crystal structures (PDB ID: 5twp and 5nj6) with sequence identities of 84.35% and 92.45% to H1-74 VH and VL regions, respectively, as modeling templates.We focused primarily on five binding regions (Figure 1C).
Binding region 1C-B's Amide-C(O)NH 2 group atop N40's side chain could form hydrogen bonds with polypeptides below, while W55, L104, and M110 could establish a hydrophobic binding pool.In binding zone 1C-C, residues F220, Y214, M110, and W52 formed a deeper binding pocket with a higher degree of hydrophobicity.
H212, located at the pocket's base, established polar rivet interactions with neighboring amino acid residues on the polypeptide bounding region.Binding region 1C-D, compact and small at the protein's top, contained highly polar R218 and D67 amino acid residues, capable of forming a strong salt bridge with associated peptides.A66, W52, F220, T64, and Y65 created shallow hydrophobic binding pockets.Binding region 1C-E consisted of multiple amino acids with distinct characteristics, including polar amino acid residues such as R216, H212, and S217, and hydrophobic amino acid residues such as Y214 and Y155.The polar amino acid residues could establish a salt bridge with strong negatively charged amino acids in polypeptides, while hydrophobic ones enabled an aromatic stacking effect by forming π-π stacking with identical amino acid residues in polypeptides through their benzene rings.

| Virtual mutation screening of polypeptide LI-6
When the peptides LPFQNI and LVLWGIHHP are combined with H1-74 they contain the same "LXXXXI" structure; this suggests a conformational epitope.To investigate mAb binding to antigenic epitope peptides with completely different structures, we mutated LPFQNI's residues into 20 different amino acid residues using Pymol software, then assessed each mutation's affinity to H1-74 via molecular docking.L307-W and I 312-Y mutations improved binding affinity and structural stability (Table 1).F309-G and N311-G mutations in LPGQGI weakened affinity to H1-74.This indicated that the mAb can bind to two antigen peptides (WPFQNY and LVLWGIHHP) with completely different structures.Finally, we obtained an artificially modified six peptide WPFQNY that can still bind to H1-74 and was completely different from the LVLWGIHHP sequence and structure, and an artificially modified six peptide LPGQGI with significantly weakened binding activity to H1-74.

| Changes in the binding characteristics of polypeptides to H1-74 mAb, pre-and postmodifications
Analysis showed LPFQNI and WPFQNY occupying six identical binding positions in H1-74's protein binding groove (Figure 2A).At binding position 1 (Figure 2B), HI-74's antibody protein was surrounded by amino acids Y65, K70, and D67, wherein the hydrophobic carbon chain hydrophobic groups on the Y65 benzene ring, K70, and D67 formed a planar hydrophobic binding region.
When bound to a polypeptide, the amino acid L side chain group on the polypeptide LPFQNI was small and the hydrophobic effect was weak.The W amino acid side chain indole group was larger and formed a stronger hydrophobic binding surface than L with the surrounding amino acids, especially the side chain phenol group on the Y65 amino acid, which was more conducive to the combination of both sides.
In the binding position 2 (Figure 2C), the amino acids bound on both polypeptides were P, and the volume of the side chain pentacion C-N heterocyclic was unchanged, so the change was less pronounced.
The position 3 region (Figure 2D) consisted of residues R218, W52, F220, H212, and Y105.Compared to F in LPFQNI, the side chain phenyl ring group of residue F on WPFQNY could be inserted    In the position 4 region (Figure 2E), the amino acids on both polypeptides were Q, and the binding patterns were not much different.
In the position 5 region (Figure 2F), the N amino acid terminal N atom on LPFQNI only formed a hydrogen bond with the O atom on L104, while the N terminal N atom on WPFQNY formed multiple hydrogen bonds with the oxygen atom on G38, the skeleton oxygen atom on L104, and the phenol group at the end of the Y105 side chain.Therefore, WPFQNY formed a stronger polar binding effect than LPFQNI in the position 5 region.
Within position 6 (Figure 2G), I only formed a hydrogen bond with W55.Y formed hydrogen bonding with N36, Y37, and G38.
Although the hydrogen bonding system was comparable, Y formed a stronger hydrophobic interaction with Y37 and Y59.Overall, when the polypeptide LPFQNI was transformed into a polypeptide WPFQNY, the binding activity of the latter to H1-74 was enhanced.

| Binding activity of polypeptides to H1-74 mAb
The binding of H1-74 to the H1N1 antigen of the influenza virus was assessed by performing blocking experiments using polypeptide LP-9, LI-6, WY-6, and LPGQGI.The binding activity between H1-74 mAb and four polypeptides was tested and verified.Based on the inhibition rate ≤ 0.4 indicated that the antibody did not recognize the epitope.The inhibition index between 0.4 and 0.8 indicated that the epitope recognized by the antibody was related to it.The inhibition rate ≥ 0.8 indicated that the antibody recognized the epitope.it was concluded that the inhibition rates of LP-9, LI-6, and WY-6 on the binding of H1-74 and HA were all above 0.8, indicating that these three peptides were recognized by H1-74.Even after the substitution of LXXXI with WXXXY in LPFQNI, it was found that WPFQNY still possessed good binding affinity to H1-74, despite having a completely different sequence from that of LVLWGIHHP (Figure 3).

| Interaction patterns of polypeptides with H1-74
To check the stability of three different systems, RMSD values were tested to monitor the conformation fluctuations of complexes over 100 ns molecular dynamics simulations (as shown in Supporting Information S1: Figure S1), all systems had reached equilibrium.
Average structure for each system was extracted from the equilibrated trajectories.To investigate the molecular basis for the cross-reaction between antibody and antigen epitope peptide binding, interaction patterns of polypeptide LP-9, LI-6, and WY-6 with the H1-74 mAb were analyzed to understand how a mAb binds to different epitopes of different antigens (Figure 4).
When LVLWGIHHP was bound to H1-74 mAb, compared with the two polypeptides LI-6 and WY-6, it could be spread more in the H1-74 antibody binding cavity, so the binding energy was the lowest (Figure 4A).L, V, and L adhered to the protein surface shallow, hydrophobic pockets, and the surrounding amino acids were expected to form a good hydrophobic interaction.This binding region consisted of amino acids W52, W55, K70, and D67, where indole groups on the W52 and W55 amino acids formed a hydrophobic matching effect.W: The indole group of its side chain was particularly large, and it was inserted deeply into the hydrophobic pocket area of the antibody, which formed an excellent hydrophobic matching effect with the hydrophobic binding pocket composed of amino acids such as W52, W55, M110, T109, Y214, N40, and so on.In particular, an aromatic stacking effect was formed with the Y214 benzene ring and the W52 indole group.G and I: The In the WPFQNY system (Figure 4C), W and P were surrounded by hydrophobic pockets formed by Y65, K70, D67, and R218, forming a good hydrophobic effect.Compared with L in LPFQNI, the tryptophan W side chain group was larger, so the hydrophobic effect of the side chain group was stronger.P: In addition to the hydrophobic interaction, hydrogen bonding with Y214 was also formed.F had good PI-PI interaction with W52 and good hydrophobic interaction with the benzene ring side chain on Y214.Compared with the F in LPFQNI, the amino acid F in WPFQNY was inserted deeper into the pocket, in addition to the hydrophobic effect described above, a new hydrophobic interaction with the amino acid F220 was formed deep in the pocket, so this part had a stronger effect.Q exhibited hydrogen bonding with N40 and G106.N exhibited hydrogen bonding with G38, L104, and Y105, of which G38 and Y105 were new hydrogen bonding systems.Y exhibited hydrogen bonding with N36, G38, and Y37.Although the hydrogen bonding system was comparable to amino acid I in the polypeptide LPFQNI, it formed a stronger hydrophobic interaction with Y37 and Y59.
It was discovered that upon binding of the H1-74 to various polypeptides, the two molecules mutually induced and conformed to each other, resulting in an optimal geometric and energetic match.

| Antipeptide serum production in peptideimmune mice
To prepare antipolypeptide serum, BALB/c mice (6-8 weeks) were immunized with LP-9, LI-6, and WY-6.The resulting serum was then tested for antibodies against H1N1 influenza virus to determine whether the polypeptides contained antigen epitopes of the influenza virus HA antigen, which could bind to H1-74 (Table 3).

Note:
The values in the table were the ratio of the optical density (OD) value detected by enzyme-linked immunosorbent assay in the experimental group to the OD value in the SP2/0 control group (the antibody and SP2/0 negative control used cell culture supernatant, and the negative control OD value was less than 0.05, it was calculated as 0.05), and the P/N ≥ 2.5 result was positive.n = 3.
Antibody cross-reaction with antigen binding is a widely observed phenomenon in immunological research.Significant strides have been made in understanding both the specific responses to antigens and antibodies, and the cross-reactivity where antibodies bind to different antigens.Current knowledge suggests that cross-reaction is often due to identical linear epitopes or similar conformational/ discontinuous epitopes on different target antigens. 17,18 bioinformatics studies of cross-reactivity epitopes, a common starting point is the search for sequence similarity.BLAST, a tool from NCBI, has been utilized to identify potential cross-reactivity epitopes in COVID-19 protein. 191][22] However, subsequent research revealed limitations to this approach; sequence similarity alone is insufficient for reliably predicting cross-reactivity epitopes between proteins.For instance, Yuan's study on a neutralizing monoclonal antibody (CR3022) demonstrated its crossreaction with the S protein of both COVID-19 and SARS.The crystal structure of CR3022 and RBD complex of two proteins was analyzed by X-ray crystal diffraction.The analysis revealed significant differences in the conformational epitopes between the COVID-19 and SARS proteins.Different binding modes between CR3022 and different epitopes on the two proteins by chemical bonds could also cross-react.This suggested that antibodies can cross-react with multiple epitopes, even those with different linear sequences or conformational structures. 23In our study, we focused on the H1-74 mAb, targeting the influenza virus HA antigen, and its binding to two specific antigenic epitope peptides, 191-LVLWGIHHP-199 and 307-LPFQNI-312. 10We combined bioinformatics analysis with the classical ELISA method to investigate the mechanism of antibody cross-reaction. 24,25 our comparison of the peptides 191-LVLWGIHHP-199 and 307-LPFQNI-312, we identified a common "LXXXXI" structure (Figure 1A), suggesting they might represent similar conformational epitopes.To explore if H1-74 mAb could bind to two distinct short peptides with different conformations, we created a new structure to replace the "LXXXXI" structure in LI-6.We performed virtual saturation mutations on LI-6 using computer molecular simulation, leading to the discovery of an affinity-enhanced mutant WY-6.This mutant's binding to H1-74 was confirmed by biological experiments (Figures 2 and 3).The ability of H1-74 to bind to two antigenic epitope peptides with completely different sequences or conformations indicates that the mAb exhibited cross-reactivity.This finding helps explain why antibodies against pathogens can bind to human tissues without similar or identical sequences or conformations. 8cent research has delved into the cross-reaction mechanism between antibodies and antigen epitopes.It was found that antibodies could induce conformational changes in target antigen epitopes, leading to allosteric transformations that make them compatible for binding. 26This implies that the geometric arrangement within some epitopes imparts cross-reactive capabilities to antibodies. 27Moreover, crystal structure analysis has shown that a single antibody can exhibit multiple specificities by binding to various antigens at different CDR regions, due to variations in ligand placement. 28,29Based on 3D structural analysis, only a small portion (20%-33%) of amino acid residues in the CDR of the antibodies were responsible for antigen binding, and this section of the amino acid residues was highly variable in each antibody molecule.A single antibody's CDR can have multiple binding recognizing different epitopes. 30,31This conformational diversity of sequences, while enhancing the antibody library's effectiveness, can also lead to autoimmunity and allergies. 32 refined our approach by excluding identical linear epitopes or similar conformational epitopes, focusing instead on the dominant foundation for analyzing the interaction patterns of H1-74 mAb with three epitope peptides LP-9, LI-6, and WY-6 through molecular simulation.Our findings highlight the complexity in mAb formation, involving hydrophobic and hydrophilic units, and groups capable of forming hydrogen bonds, salt bonds, and π-π stacking (Figure 4).We pinpointed the locations of these structures.The interaction process begins with collisions between groups on the antibody and antigen epitope, leading to molecular binding as the sum of various forces reaches a certain threshold.After that, a bondage was formed between the two molecules.Importantly, the antibodies did not distinguish between molecules based on their origin or sequence variance.
The binding affinity of H1-74 was influenced by diverse functional units or combinations of key amino acid residues within its CDR, as shown in Table 2.This variability forms the basis for antibody cross-reaction.The hydrophobic effects were more pronounced between WPFQNY and H1-74 when compared to LPFQNI, which was attributed to the greater binding surface area and the larger W side chain group, in contrast to the small L size.In addition, amino acid I formed a hydrogen bond with W55, while Y formed a more intensive hydrogen bond system with residues N36, Y37, and G38 on H1-74.Stronger hydrophobic interactions were also formed between residues Y37 and Y59 on H1-74 and peptide WPFQNY (Figure 2).
In Table 2, the amino acid residues in bold represent those readily binding to epitopes in the static state of the variable region of the antibody.The italicized residues, such as K70, T109, T35, Y59, T60, G38, and G106, are either hidden within the protein structure or distant from the antigen binding region, and remained unaffected by the static state of the antibody.The antibody and polypeptide, which were mutually induced, initially bound together, leading to the exposure of protein allosteric or groups and their subsequent proximity to each other. 26,32Y214 was the only amino acid among five of the antigen-binding region E that facilitated binding in the antibody's static state.The remaining residues, S217, R216, H212, and Y155, while "prepared" for binding to the antigen, were not utilized in binding to these three polypeptides, suggesting H1-74's potential to bind to more antigen epitopes.
yielded three mAbs that recognized two distinct sequences on HA antigens.Influenza viruses are categorized into four types: A, B, C, and D, with type A being the most severe.The 1918 influenza A (H1N1) outbreak in Spain and the 2009 H1N1 outbreak in Mexico had significant societal impacts. 11This study concentrated on the H1-74 mAb produced by the 2009 H1N1 influenza virus HA protein, which reacts with two peptides, LVLWGIHHP (191aa-199aa) and LPFQNI (307aa-312aa), derived from the HA protein.We aimed to investigate the molecular mechanism of antibody cross-reaction and determine if varying combinations of key amino acid residues within the complementary determining region (CDR) of antibody molecules could facilitate cross-reaction.This study also sought to clarify the link between pathogenic microbial infection and autoimmune disease, even in the absence of homologous sequences or similar conformational epitopes in cross-reacting molecules.Early removal of pathogenic microorganisms in patients to prevent the production of such cross-reactive antibodies may offer a new approach to treating autoimmune diseases.

F
I G U R E 1 Simulation structure of H1-74 mAb variable region formation of hydrophobic, hydrophilic pockets which may form salt bonds, hydrogen bonds, and so on.(A) The positions of LP-9 and LI-6 on the three-dimensional structure of hemagglutinin (HA).Red represented LP-9 and purple represented LI-6.(B) The variable region gene of light and heavy chain of H1-74 mAb was cloned and sequenced.(C) The H1-74 mAb consisted of light and heavy chains.One long antigen-binding pocket was formed by 8 and 16 amino acids from the H1-74 VL and H1-74 VH subunit, respectively.To ensure one more intuitive view of binding sites, five polypeptide-binding regions of binding pocket on the H1-74 protein (region A-E) were divided based on the geometric shapes and physicochemical properties.
T A B L E 1 The binding activity of different amino acid mutants of LPFQNI polypeptide with H1-74 mAb.

F I G U R E 2
Variable region structure docking analysis of LPFQNI, WPFQNY, and H1-74.Binding modes of peptides LPFQNI (colored green) and WPFQNY (colored magenta) to the protein H1-74.The binding surface was shown as white surface.Residues forming the binding interface within 4.5 Å from each other between peptides and H1-74 were shown as sticks.Hydrogen bonds were shown as black dashes.(A) The peptides LPFQNI and WPFQNY occupied the identical six binding positions in the H1-74 protein binding groove.(B-G) Binding modes difference of peptides LPFQNI and WPFQNY to the H1-74 at six binding positions 1-6.deeper into the hydrophobic binding pocket.A better hydrophobic matching effect was formed between the indole hydrophobic planar group on the W52 amino acid and the benzene ring on the Y105.In addition to the hydrophobic effect, amino acid F on WPFQNY also formed a new hydrophobic interaction with the amino acid F220 side chain benzene ring deep in the pocket, so that the WPFQNY polypeptide bound to H-74, and the hydrophobic effect in the position 2 region was stronger than LPFQNI.
former skeleton C( = O)-N and the lateral side chain carbon chain, between the surrounding amino acids, especially W55 and Y37, formed a good hydrophobic compensation effect.H and H: amino acids formed neutral polar interactions with nearby amino acids, qualitatively reflected in hydrogen bonding systems.These hydrogen bonds included histidine H-N (nitrogen)…O (oxygen)-Y105 and histidine H-N (nitrogen)…O (oxygen)-N36.P: The oxygen atoms of the terminal skeleton formed a good hydrogen bond with the N atoms on N57, and the hydrophobic interaction between the carbon and nitrogen pentaary rang on the side chain and the surrounding amino acids was formed.In the LPFQNI system (Figure4B), L and P could adhere to the shallow surface of the protein, hydrophobic pockets, forming a good hydrophobic interaction.The amino acids R218, D67, K70, A66, T64, F I G U R E 3 Inhibition rate of the reaction of original and mutant peptides with H1-74.Y214, and W52 and polypeptides, especially between P and I, formed an excellent hydrophobic matching effect on the H1-74 protein.In addition, the oxygen atoms in the proline P backbone could form hydrogen bound with phenol on Y214.F: The sidechain phenyl cyclic group was a hydrophobic chemical group, which was inserted into the hydrophobic binding pocket composed of amino acids such as W52 and Y214 on the insertion protein, forming a strong hydrophobic interaction pattern.Q and N: The skeleton C( = O)-N formed a good hydrophilic compensation effected with the surrounding amino acids, including glutamine Q-N (nitrogen)…O(oxygen)-Y214, glutamine Q-O(oxygen)… N (nitrogen)-N40 and asparagine N-N(nitrogen)…O (oxygen)-L104.I: C atoms on amino acid side chain groups formed a good hydrophobic interaction with the surrounding hydrophobic environment.

F I G U R E 4 4
Binding pattern of H1-74 mAb with polypeptide LP-9, LI-6, and WY-6.(A-C) Schematic of binding interface between peptides and H1-74 mAb.Binding interface regions for peptides and H1-74 mAb were colored as follows: H1-74 in white and peptides in green.Key residues forming binding interface were shown as sticks and colored as white and green.The hydrogen bonds were shown in dotted lines.(D-F) Hydrophpbic and hydrophilic binding surfaces for H1-74 and peptides complexes.T A B L E 2The bond energy of H1-74 mAb binding to each immunodominant group on peptide LP-9, LI-6, and WY-6.Identification of reaction characteristics of anti-LP-9 mAbs with three polypeptides and influenza virus antigens.