Related parameters of affinity and stability prediction of HLA-A*2402 restricted antigen peptides based on molecular docking.

BACKGROUND: Major histocompatibility complex class I (MHC-I) plays an important role in cell immune response, and stable interaction between polypeptides and MHC-I ensures efficient presentation of polypeptide-MHC-I (pMHC-I) molecular complexes to T cells. The aim of this study was to explore ways to improve the affinity and stability of the p-Human Leukocyte Antigen (HLA)-A*2402 complex.
METHODS: The peptide sequences of the restricted antigen peptides for HLA-A*2402 and the results of the in vitro competitive binding test were retrieved from the literature. The affinity values were predicted using NetMHCpan v4.1 server, and the stability values were predicted using the NetMHCstab v1.0 server. Auto Vina was used to dock peptides to HLA-A*2402 protein in a flexible docking manner, while Flexpepdock was employed to optimize the docking morphology. Maestro was used to analyze the intermolecular forces and the binding affinity of the complex, while MM-GBSA was used to calculate the binding free energy values.
RESULTS: The intermolecular interactions that maintained the affinity and stability of peptide-HLA-A*2402 complex relied mainly on HB, followed by pi stack. The binding affinity values of molecular docking were associated with the predicted values of affinity and stability, the binding affinity and the binding free energy, as well as the intermolecular force pi-stack. The pi stack had a significant negative correlation with binding affinity and binding free energy. The replacement of the residues of the polypeptides that did not form pi-stack interactions with HLA-A*2402 improved the affinity and/or stability compared to before replacement.
CONCLUSIONS: The generation and increase in the number of pi-stacks between peptides and HLA-A*2402 molecules may help improve the affinity and stability of p-HLA-A*2402 complexes. The prediction of intermolecular forces and binding affinity of peptide-HLA by means of molecular docking is a supplement to the current commonly used prediction databases. 2021 Annals of Translational Medicine. All rights reserved.

Introduction

Major histocompatibility complex class I (MHC-I) molecules plays an important role in the cellular immune response, presenting peptides to cytotoxic T lymphocytes (CTL) and allowing the immune system to carefully examine ongoing biological processes within the cell (1). Many studies on immunotherapy have found that tumor-specific antigen peptides not only bind to MHC by means of low affinity, but also often exhibit function defects in antigen processing and presentation, leading to immune evasion (2). This poses a huge challenge to T cell-based immunotherapy. HLA-A plays an important role in anti-tumor immune response and tumor neoantigen discovery, and HLA-A*24 is an allelic type of HLA-A (3,4). HLA-A*2402 is one of the most common alleles in East Asian populations, especially in Japanese and Chinese populations (5,6). Recent studies on HLA-A*2402 have focused on the clinical application of HLA-A*2402 restricted antigen. In a study on the safety, immune response rate and clinical benefit of cancer vaccine combined with chemotherapy, it was found that patients with HLA-A*2402 positive, locally advanced, metastatic and/or recurrent gastrointestinal, lung or cervical cancer, their specific T cell response rate of HLA-A*2402 restricted tumor-associated epitope peptide was significantly correlated with longer overall survival (7). In another study of new vaccine therapy evaluating HLA-A*2402 positive recurrent/progressive high-grade glioma patients using a variety of glioma cancer antigens and glioma angiogenesis-related antigen peptides, was found that this therapy was well tolerated, without any serious systemic adverse events, and could induce a strong antigen peptide-specific T lymphocyte response (8). However, the above-mentioned studies only used a variety of HLA-A*2402 restricted antigen peptides in combination with other therapeutic methods for anti-tumor therapy, and did not further explore how to replace HLA-A*2402 residues through molecular simulations, in order to better improve their roles in the anti-tumor immune response. Increasing the complementarity between the binding clefts of peptides and HLA-A molecules by replacing HLA anchor residues was a common step to improve the binding capacity and antigenicity of antigen peptides (9,10). However, this change must be based on the allelic types of each HLA-A molecule and may recruit different specific CTLs due to the conformational change of the antigen peptide, thereby reducing the recognition probability of T cells (11). The efficient presentation of polypeptide-MHC-I class (pMHC-I) molecular complexes to T cells benefited from the stable interaction between polypeptides and MHC-I (12). Compared with affinity, the stability of the pMHC-I complex could better reflect the immunogenicity of CTL (13), but it was difficult to distinguish stability from other elements of MHC-I binding, such as affinity. In recent years, scientists’ interest in artificial neural networks (ANN) has increased day by day. It is a rough simulation of the information processing capabilities of the human brain. It is a modern and complex computing technology that plays a huge role in drug analysis, drug technology, and screening of new drugs (14,15). Scientists have established a high-throughput method for evaluating the stability of the pMHC-I complexes using an ANN method to predict the half-life of pMHC-I complex binding (16). There are two novel tools that identify with relatively high accuracy. The two tools consist of (I) the NetMHCpan-4.1 server predicts binding of peptides to any MHC molecule of known sequence (17), and (II) NetMHCstab-1.0 predicts the stability of peptide binding to a number of different MHC molecules (18). Researches on molecular docking of protein-peptide interactions are difficult and time-consuming tasks because peptides are generally more flexible than proteins and tend to adopt multiple conformations. In the process of searching for binding sites for peptides, both peptide and protein molecules have significant conformational flexibility (19,20). At present, using the flexible molecular docking method in virtual screening to predict the binding affinity of polypeptides with different MHC allotypes has proven to have a fairly high prediction accuracy (21). The intent of the present study was to understand the intermolecular force, binding affinity, binding energy, affinity predicted values, and stability predicted values of HLA-A*2402 with restricted antigen peptides. In addition, we further analyzed the results of the in vitro competitive binding test, as well as the correlation between other parameters, and explored ways to improve the stability of the p-HLA-A*2402 complex.

Methods

Data collection

The polypeptide sequences of the HLA-A*2402 restricted antigen peptide and the results of the in vitro competitive binding test were obtained from the literature (22) and the affinity between the antigen peptide and HLA-A*2402 was predicted by the NetMHCpan v4.1 server (http://www.cbs.dtu.dk/services/NetMHCpan/) (17). The stability values of peptides and HLA-A*2402 were predicted by NetMHCstab v1.0 server (http://www.cbs.dtu.dk/services/NetMHCstab/) (18).

Molecular docking and dynamic simulation

The crystal model of the peptide-HLA-A*2402 complex (PDB ID: 2BCK) was obtained from the PDB database (http://www.rcsb.org/) (23,24) and Auto Vina (25) was used to dock the HLA-A*2402 restricted antigen peptide to the HLA-A*2402 protein in a flexible docking manner. Maestro (Schrodinger, LLC, New York, NY, 2019) (26) analyzed the intermolecular force of the complex [hydrogen bond (HB) and pi-stack] and the binding affinity values of the complex, while Flexpepdock (27) further optimized the docking morphology of the restricted antigen peptides and HLA-A*2402, and analyzed the binding affinity values of the complex. MM-GBSA (28) calculated the binding free energy values of HLA-A*2402-restricted antigen peptide. The above process was performed under the premise that the parameters of each docking system and the kinetic simulation were consistent.

Analysis of relevant parameters

The previous correlations of various parameters such as affinity prediction values, stability prediction values, intermolecular force, binding affinity, binding free energy, and in vitro competitive binding test results were analyzed and further explored the way to improve the affinity and stability of HLA-A*2402 with the restricted antigen peptides.

Statistical analysis

The correlation analysis used Spearman correlation coefficient statistical analysis, and P<0.05 was considered statistically significant.

Results

Relationship between the in vitro competitive binding capacity of peptides and the predicted values of affinity and stability

The sequences of HLA-A*2402 restricted antigen peptides, the results of the peptide in vitro competitive binding tests, the affinity predicted values of NetMHCpan v4.0, and the stability predicted values of NetMHCstab v1.0 are shown in .

Table 1

Peptide sequences, in vitro binding capacity, affinity, and stability prediction values

No.	Peptide sequences	In vitro binding capacity (IC50, nM)	% Rank			Thalf(h)
			Values	Binding		Values	Stability
1	CDSTLRLCV	0	48	NB		0.28	NS
2	CYEQFNDSS	0	23	NB		0.39	NS
3	CYEQLNDSS	0	25.917	NB		0.38	NS
4	CYSLYGTTL	0.10	0.364	SB		2.67	WS
5	CYSVYGTTL	0.24	0.346	SB		2.61	WS
6	DFAFRDLCI	3.27	4.653	NB		0.36	NS
7	DKKQRFHNI	55.95	13.036	NB		0.34	NS
8	DPQERPRKL	0	9.624	NB		0.28	NS
9	EYMLDLQPE	0	13.964	NB		0.46	NS
10	EYRHYCYSL	0.32	0.873	WB		0.73	NS
11	EYRHYCYSV	8.64	2.866	NB		0.56	NS
12	FYSKISEYR	0	3.002	NB		0.85	NS
13	HYCYSVYGT	0	9.818	NB		0.89	NS
14	HYNIVTFCC	6.06	3.974	NB		1.95	NS
15	KCLKFYSKI	7.50	6.878	NB		0.79	NS
16	KFYSKISEY	41.70	0.851	WB		0.42	NS
17	KKQRFHNIR	28.64	37	NB		0.29	NS
18	KLPQLCTEL	0.83	2.258	NB		0.43	NS
19	LLRREVYDF	0	4.462	NB		0.35	NS
20	LQTTIHDII	1.42	7.86	NB		0.36	NS
21	LQTTIHEII	1.65	5.813	NB		0.37	NS
22	LYCYEQFND	0	21.5	NB		0.72	NS
23	LYGTTLEQQ	0	12	NB		0.68	NS
24	PYAVCDKCL	1.97	2.942	NB		0.48	NS
25	QYNKPLCDL	2.97	0.742	WB		0.79	NS
26	RAHYNIVTF	0.15	0.814	WB		0.52	NS
27	RCINCQKPL	0	37.667	NB		0.31	NS
28	RFHNIRGRW	23.93	0.728	WB		0.5	NS
29	RHLDKKQRF	0	0.718	WB		0.61	NS
30	RWTGRCMSC	0	6.024	NB		0.37	NS
31	TDLYCYEQF	0	5.845	NB		0.75	NS
32	TTLEQQYNK	0	16.818	NB		0.33	NS
33	VDIRTLEDL	0	16.436	NB		0.35	NS
34	VYCKQQLLR	46.40	4.794	NB		1.11	NS
35	VYDFAFRDL	0.10	0.435	SB		0.54	NS
36	VYGTTLEQQ	0	7.977	NB		0.74	NS

SB, strong binding; NB, no binding; WB, weak binding, NS, unstable; WS, weak stability; HS, strong stability.

Among these, the threshold of affinity prediction: the threshold of strong binding prediction: %Rank ≤0.5 was identified as strong binding (SB), the threshold of weak binding prediction: 0.5< %Rank ≤2 (WB), and the rest of the values were identified as no binding (NB). Stability prediction threshold: strong stability (HS) prediction threshold (hours): Thalf(h) ≥6, weak stability (WS) prediction threshold (hours): 2≤ Thalf(h) <6, and other values were identified as unstable (NS). As shown in , 19 of the 36 antigen polypeptides were competitively binding with HLA-A*2402 molecules in vitro, and among these, three were predicted to have strong binding strength, five peptides were predicted to have weak binding capacity, and the remaining 11 were predicted to have no binding capacity. However, the remaining 17 polypeptides had no binding ability. Among the peptides with both in vitro competitive binding ability and predicted affinity, only two were predicted to have weak stability, namely CYSLYGTTL and CYSVYGTTL, respectively.

Intermolecular force and binding energy values of the peptide- HLA-A*2402 complex

The intermolecular forces (HB and pi-stack), binding affinity, and binding free energy values of the peptide-HLA-A*2402 complex are shown in . The intermolecular interaction that maintained the polypeptide-HLA-A*2402 complex are also shown in , and were mainly based on HB, followed by pi-stack.

Table 2

Intermolecular force and binding energy values of the peptide-HLA-A*2402 complex

No.	Peptide sequences	Binding affinity of Auto Vina (kcal/mol)	Binding affinity of Flexpepdock (kcal/mol)	Binding free energy of MM-GBSA (kJ/mol)	Numbers of HB	Numbers of pi-stack
1	CDSTLRLCV	−7.4	−287.789	−76.82	7	0
2	CYEQFNDSS	−8.7	−290.077	−58.71	11	0
3	CYEQLNDSS	−8.1	−284.597	−41.36	6	2
4	CYSLYGTTL	−8.3	−289.04	−60.66	7	0
5	CYSVYGTTL	−8.6	−289.87	−71.87	6	2
6	DFAFRDLCI	−9.3	−281.64	−108.11	12	0
7	DKKQRFHNI	−7.8	−288.634	−43.97	7	0
8	DPQERPRKL	−8.1	−286.014	−73.64	6	0
9	EYMLDLQPE	−9.4	−300.616	−128.51	13	2
10	EYRHYCYSL	−9.6	−297.273	−130	10	3
11	EYRHYCYSV	−9.6	−299.058	−85.96	9	1
12	FYSKISEYR	−9.3	−291.428	−142.82	7	0
13	HYCYSVYGT	−9.5	−298.288	−98.3	6	5
14	HYNIVTFCC	−9.9	−297.126	−123.69	6	0
15	KCLKFYSKI	−7.7	−286.985	−121.96	8	0
16	KFYSKISEY	−9.2	−297.436	−141.45	11	0
17	KKQRFHNIR	−8.2	−293.142	−91.23	4	0
18	KLPQLCTEL	−8.1	−284.883	−71.18	5	0
19	LLRREVYDF	−9.4	−297.278	−154.01	9	1
20	LQTTIHDII	−8.5	−299.078	−69.67	5	0
21	LQTTIHEII	−8.6	−292.953	−109.99	10	0
22	LYCYEQFND	−9.9	−281.177	−106.04	7	0
23	LYGTTLEQQ	−8.7	−294.78	−135.25	12	2
24	PYAVCDKCL	−9.6	−291.84	−96.25	7	2
25	QYNKPLCDL	−9.3	−285.88	−75.06	5	0
26	RAHYNIVTF	−10	−294.023	−120.77	7	1
27	RCINCQKPL	−8.5	−286.983	−72.71	7	0
28	RFHNIRGRW	−9.9	−299.645	−160.01	9	2
29	RHLDKKQRF	−8.6	−288.879	−88.11	6	0
30	RWTGRCMSC	−7.8	−284.186	−146.42	10	2
31	TDLYCYEQF	−9	−293.73	−99.91	11	0
32	TTLEQQYNK	−9	−286.907	−38.65	3	0
33	VDIRTLEDL	−8.8	−300.057	−90.65	9	0
34	VYCKQQLLR	−8.9	−289.743	−124.19	8	2
35	VYDFAFRDL	−10.4	−295.182	−134.11	12	2
36	VYGTTLEQQ	−9.2	−289.335	−66.4	4	0

The correlation statistical analysis results of the in vitro competitive binding capacity (experimental binding capacity), the numbers of HB, the numbers of pi-stacks, the Auto binding capacity, the Flex binding capacity, the binding energy, the affinity prediction values (%Rank), and the stability prediction values [Thalf(h)] are shown in , where * represented P<0.05, and ** represented P<0.01. show the results of correlation analysis among the Experimental binding capacity (EBC), the values of %Rank and Thalf(h), the Auto binding affinity, the Flex binding affinity, the GBSA binding free energy, and the numbers of HB and pi-stacks. The Spearman correlation coefficient was applied to indicate the strength of the correlation.

Table 3

Correlation of in vitro competitive binding capacity with predicted values, binding affinity, and binding free energy

Parameters	Correlation analysis	EBC
%Rank	Correlation coefficient	−0.390*
	P value	0.019
Thalf(h)	Correlation coefficient	0.122
	P value	0.479
Auto	Correlation coefficient	−0.08
	P value	0.645
Flex	Correlation coefficient	−0.143
	P value	0.405
GBSA	Correlation coefficient	−0.123
	P value	0.475
HB	Correlation coefficient	0.001
	P value	0.993
pi-stack	Correlation coefficient	−0.018
	P value	0.917

*, P<0.05.

Table 4

Correlation of predicted affinity with competitive binding in vitro, binding affinity, and binding free energy

Parameters	Correlation analysis	%Rank
Thalf(h)	Correlation coefficient	−0.551**
	P value	0.001
EBC	Correlation coefficient	−0.390*
	P value	0.019
Auto	Correlation coefficient	0.394*
	P value	0.018
Flex	Correlation coefficient	0.19
	P value	0.266
GBSA	Correlation coefficient	0.309
	P value	0.067
HB	Correlation coefficient	−0.088
	P value	0.609
pi-stack	Correlation coefficient	−0.22
	P value	0.197

*, P<0.05; **, P<0.01.

Table 5

Correlation of stability prediction values with in vitro competitive binding capacity, binding affinity, and binding free energy

Parameters	Correlation analysis	Thalf (h)
EBC	Correlation coefficient	0.122
	P value	0.479
%Rank	Correlation coefficient	−0.551**
	P value	0.001
Auto	Correlation coefficient	−0.379*
	P value	0.023
Flex	Correlation coefficient	−0.126
	P value	0.464
GBSA	Correlation coefficient	−0.21
	P value	0.218
HB	Correlation coefficient	0.001
	P value	0.995
pi-stack	Correlation coefficient	0.265
	P value	0.118

*, P<0.05; **, P<0.01.

Table 6

Correlation of binding affinity by Auto with predicted values, in vitro competitive binding capacity, binding affinity, and binding free energy

Parameters	Correlation analysis	Auto
EBC	Correlation coefficient	−0.08
	P value	0.645
Thalf(h)	Correlation coefficient	−0.379*
	P value	0.023
%Rank	Correlation coefficient	0.394*
	P value	0.018
Flex	Correlation coefficient	0.476**
	P value	0.003
GBSA	Correlation coefficient	0.462**
	P value	0.005
HB	Correlation coefficient	−0.244
	P value	0.151
pi-stack	Correlation coefficient	−0.365*
	P value	0.029

*, P<0.05; **, P<0.01.

Table 7

Correlation of binding affinity by Flex with predicted values, in vitro competitive binding capacity, binding affinity, and binding free energy

Parameters	Correlation analysis	Flex
EBC	Correlation coefficient	−0.143
	P value	0.405
Thalf(h)	Correlation coefficient	−0.126
	P value	0.464
%Rank	Correlation coefficient	0.19
	P value	0.266
Auto	Correlation coefficient	0.476**
	P value	0.003
GBSA	Correlation coefficient	0.403*
	P value	0.015
HB	Correlation coefficient	−0.326
	P value	0.052
pi-stack	Correlation coefficient	−0.345*
	P value	0.039

*, P<0.05; **, P<0.01.

Table 8

Correlation of binding free energy, predicted values, in vitro competitive binding capacity, binding affinity, and binding free energy

Parameters	Correlation analysis	GBSA
EBC	Correlation coefficient	−0.123
	P value	0.475
Thalf(h)	Correlation coefficient	−0.21
	P value	0.218
%Rank	Correlation coefficient	0.309
	P value	0.067
Auto	Correlation coefficient	0.462**
	P value	0.005
Flex	Correlation coefficient	0.403*
	P value	0.015
HB	Correlation coefficient	−0.601**
	P value	0
pi-stack	Correlation coefficient	−0.407*
	P value	0.014

*, P<0.05; **, P<0.01.

Table 9

Correlation of the numbers of HB with predicted values, in vitro competitive binding capacity, binding affinity, and binding free energy

Parameters	Correlation analysis	HB
EBC	Correlation coefficient	0.001
	P value	0.993
Thalf(h)	Correlation coefficient	0.001
	P value	0.995
%Rank	Correlation coefficient	−0.088
	P value	0.609
Auto	Correlation coefficient	−0.244
	P value	0.151
Flex	Correlation coefficient	−0.326
	P value	0.052
GBSA	Correlation coefficient	−0.601**
	P value	0
pi-stack	Correlation coefficient	0.298
	P value	0.077

**, P<0.01.

Table 10

Correlation of the numbers of pi-stacks with predicted values, in vitro competitive binding capacity, binding affinity, and binding free energy

Parameters	Correlation analysis	pi-stack
EBC	Correlation coefficient	−0.018
	P value	0.917
Thalf(h)	Correlation coefficient	0.265
	P value	0.118
%Rank	Correlation coefficient	−0.22
	P value	0.197
Auto	Correlation coefficient	−0.365*
	P value	0.029
Flex	Correlation coefficient	−0.345*
	P value	0.039
HB	Correlation coefficient	0.298
	P value	0.077
GBSA	Correlation coefficient	−0.407*
	P value	0.014

*, P<0.05.

shows that the correlation coefficient between EBC and %Rank was 0.623, and had a significant level of 0.01, indicating that there was a significant negative correlation between EBC and %Rank (P<0.05). However, there was no correlation among EBC and Thalf(h), Auto, Flex, and GBSA, numbers of HB and pi-stacks. indicates that %Rank had a significant negative correlation with Thalf(h) and EBC (P<0.05), and a significant positive correlation with Auto (P<0.05), although there was no correlation among %Rank and Flex, GBSA, and the numbers of HB and pi-stacks. shows that Thalf(h) had a significant negative correlation with %Rank and Auto (P<0.05), but there was no correlation among Thalf(h), EBC, Flex, GBSA, and the numbers of HB and pi-stacks. shows that Auto had a significant positive correlation with %Rank, Flex, and GBSA (P<0.05), and a significant negative correlation with Thalf(h) and the numbers of pi-stacks (P<0.05). However, there was no correlation among Auto, EBC, and the numbers of HB. shows Flex had a significant positive correlation with Auto and GBSA (P<0.05), and a significant negative correlation with the number of pi-stacks (P<0.05), but no correlation among Flex, EBC, Thalf(h), %Rank, and the numbers of HB seen. shows that while GBSA had a significant positive correlation with Auto and Flex (P<0.05), and a significant negative correlation with the numbers of pi-stacks and HB (P<0.05), there was no correlation among GBSA, EBC, Thalf(h), and %Rank. shows there is a significant negative correlation between the numbers of HB and GBSA (P<0.05). However, there was no correlation among the numbers of HB, EBC, Thalf(h), %Rank, Auto, Flex, and the numbers of pi-stacks. Finally, shows there is a significant negative correlation among the number of pi-stacks, Auto, Flex, and GBSA (P<0.05), but no correlation among the numbers of pi- stacks, EBC, Thalf(h), %Rank, and the numbers of HB.

Binding affinity by Auto was related to the predicted values of affinity and stability, binding affinity by Flex, and binding free energy. Moreover, it also closely related to the intermolecular force pi-stack. There was a significant negative correlation among the numbers of pi-stacks, binding affinity, and binding free energy. This suggests that the numbers of pi-stacks played an important role in the interaction of peptides and HLA-A*2402. Furthermore, the amino acid residues that form the pi-stack interaction between the polypeptide and HLA-A*2402 were screened, as shown in , which also shows P1-P9 represented the amino acid residues 1 to 9 of the polypeptide, respectively, and A represented HLA-A*2402. The results also indicate that pi-stacks were mainly composed of Y (Tyr) on the polypeptide, which was mainly located at position 2, 4, 5, and 7, H (His) that was mainly located at position 1 and 3, W (Trp) that was located at position 2 and 9, and F (Phe) that was located at position 9. However, the residues of position 6 and 8 of polypeptides did not form a pi-stack with HLA-A*2402. The residues on HLA-A*2402 that formed pi-stacks with those on the polypeptide were mainly Y (Tyr) at positions 7, 116, and 123, H (Hie) at position 70, F (Phe) at position 99, and W (Trp) at position 147. It can also be seen that the interaction of pi-stacks mainly occurred among residues Y, H, W, and F, and H was a positively charged basic amino acid, while Y, W, and F were all aromatic amino acids.

Table 11

Residues of peptides and HLA-A*2402 formed pi-stack interaction

No.	Peptide sequences	In vitro competitive binding capacity	P1	P2	P3	P4	P5	P6	P7	P8	P9
1	CYEQLNDSS	NB	None	1x pi-stack to A:7: Tyr. 1x pi-stack to A:99: Phe	None	None	None	None	None	None	None
2	CYSVYGTTL	B	None	1x pi-stack to A:7: Tyr. 1x pi-stack to A:99: Phe	None	None	None	None	None	None	None
3	EYMLDLQPE	NB	None	1x pi-stack to A:7: Tyr. 1x pi-stack to A:99: Phe	None	None	None	None	None	None	None
4	EYRHYCYSL	B	None	None	None	None	1x pi-stack to A:70: Hie	None	2x pi-stack to A:147: Trp	None	None
5	EYRHYCYSV	B	None	None	None	None	None	None	1x pi-stack to A:147: Trp	None	None
6	HYCYSVYGT	NB	1x pi stack to A:7: Tyr 1x pi stack to A:99: Phe	None	None	1x pi-stack to A:99: Phe	None	None	2x pi -tack to A:147: Trp	None	None
7	LLRREVYDF	NB	None	None	None	None	None	None	None	None	1x pi-stack to A:116: Tyr
8	LYGTTLEQQ	NB	None	1x pi-stack to A:7: Tyr. 1x pi-stack to A:99: Phe	None	None	None	None	None	None	None
9	PYAVCDKCL	B	None	1x pi-stack to A:7: Tyr. 1x pi-stack to A:99: Phe	None	None	None	None	None	None	None
10	RAHYNIVTF	B	None	None	1x pi-stack to A:7: Tyr	None	None	None	None	None	None
11	RFHNIRGRW	B	None	None	None	None	None	None	None	None	1x pi stack to A:116: Tyr 1x pi stack to A:123: Tyr
12	RWTGRCMSC	NB	None	2x pi-stack to A:99: Phe	None	None	None	None	None	None	None
13	VYCKQQLLR	B	None	1x pi-stack to A:7: Tyr. 1x pi-stack to A:99: Phe	None	None	None	None	None	None	None
14	VYDFAFRDL	B	None	1x pi-stack to A:7: Tyr. 1x pi-stack to A:99: Phe	None	None	None	None	None	None	None

Residues replacement, and affinity and stability values prediction

Certain residues on polypeptides that did not form pi-stacks with HLA-A*2402 were replaced as follows: replacing the non-Y (Tyr) at position 2, 4, 5, and 7 of polypeptide with Tyr; the non-H (His) at position 1 and 3 with His; the non-W (Trp) at position 2 and 9 with Trp; and the non-F (Phe) at position 9 with Phe. shows the predicted values of affinity and stability of the polypeptide before and after residue replacement with HLA-A*2402. It is seen that after the residue replacement, of the 22 polypeptides that did not produce intermolecular pi-stack interaction with HLA-A*2402 before, 20 polypeptides had improved affinity and/or stability after the residue replacement. In addition, when excluding the residue substitution sites with affinity and/or stability reduced or unchanged, it was found that the residue sites that mainly occurred at position 2 (C2Y, F2Y, Q2Y, H2Y, L2Y, P2Y, T2Y, D2W, Q2W, H2W, L2W, P2W), position 4 (I4Y), position 7 (T7Y, C7Y, Q7Y), and position 9 (C9F, D9F, K9F, Q9F, S9F, Y9F, C9W, D9W, K9W, Q9W, S9W, Y9W). This means the residues (C, D, Q, H, L, P, T) at position 2 on the polypeptide were replaced by Y and W, the residues (I) at position 4 were replaced by Y, the residues (T, C, Q) at position 7 were replaced by Y, and the residues (C, D, K, Q, S, Y) at position 9 were replaced by F and W, and the predicted affinity and/or stability values of the polypeptide after the replacement were all higher than before. At the same time, it was found that the predicted strong/weak affinity between HLA-A*2402 and the peptide did not necessarily mean that they had strong/weak stability, while the complexes predicted to have strong/weak stability must have strong/weak affinity between the molecules.

Table 12

Relationship of peptide residue substitution, and affinity and stability values prediction

No.	Residue substitution (before replacement-position-after replacement)	Peptide sequences	Affinity values prediction	Binding	Stability values prediction	Stability
1	Original	CYEQFNDSS	23	NB	0.39	NS
	S9F	CYEQFNDSF	0.403	SB	2.08	WS
	S9W	CYEQFNDSW	0.683	WB	1.42	NS
2	Original	CYSLYGTTL	0.364	SB	2.67	WS
	T7Y	CYSLYGYTL	0.249	SB	6.34	HS
	L9F	CYSLYGTTF	0.099	SB	6.68	HS
3	Original	DFAFRDLCI	4.653	NB	0.36	NS
	F2Y	DYAFRDLCI	1.705	WB	0.69	NS
	I9F	DFAFRDLCF	1.315	WB	0.46	NS
	I9W	DFAFRDLCW	1.87	WB	0.39	NS
4	Original	DKKQRFHNI	13.036	NB	0.34	NS
	K2Y	DYKQRFHNI	0.405	SB	2.19	WS
	K2W	DWKQRFHNI	1.013	WB	0.61	NS
5	Original	DPQERPRKL	9.624	NB	0.28	NS
	P2Y	DYQERPRKL	0.452	SB	0.9	NS
	P2W	DWQERPRKL	1.104	WB	0.4	NS
6	Original	FYSKISEYR	3.002	NB	0.85	NS
	R9F	FYSKISEYF	0.004	SB	10.53	HS
	R9W	FYSKISEYW	0.017	SB	7.49	HS
7	Original	HYNIVTFCC	3.974	NB	1.95	NS
	I4Y	HYNYVTFCC	3.475	NB	2.32	WS
	F7Y	HYNIVTYCC	3.296	NB	2.12	WS
	C9F	HYNIVTFCF	0.058	SB	11.86	HS
	C9W	HYNIVTFCW	0.159	SB	8.7	HS
8	Original	KCLKFYSKI	6.878	NB	0.79	NS
	C2Y	KYLKFYSKI	0.032	SB	13.92	HS
	I9F	KCLKFYSKF	1.62	WB	1.19	NS
	C2W	KWLKFYSKI	0.283	SB	3.15	WS
9	Original	KFYSKISEY	0.851	WB	0.42	NS
	F2Y	KYYSKISEY	0.195	SB	1	NS
	S7Y	KFYSKIYEY	0.378	SB	0.58	NS
	Y9F	KFYSKISEF	0.039	SB	1.42	NS
	Y9W	KFYSKISEW	0.108	SB	1	NS
10	Original	KLPQLCTEL	2.258	NB	0.43	NS
	L2Y	KYPQLCTEL	0.1	SB	4.56	WS
	L5Y	KLPQYCTEL	1.947	WB	0.46	NS
	T7Y	KLPQLCYEL	1.225	WB	0.63	NS
	L9F	KLPQLCTEF	0.347	SB	0.67	NS
	L2W	KWPQLCTEL	0.45	SB	1	NS
	L9W	KLPQLCTEW	0.494	SB	0.54	NS
11	Original	LQTTIHDII	7.86	NB	0.36	NS
	Q2Y	LYTTIHDII	0.38	SB	2.51	WS
	I9F	LQTTIHDIF	1.92	WB	0.47	NS
	Q2W	LWTTIHDII	1.385	WB	0.72	NS
12	Original	LQTTIHEII	5.813	NB	0.37	NS
	Q2Y	LYTTIHEII	0.184	SB	3.02	WS
	I9F	LQTTIHEIF	1.272	WB	0.49	NS
	Q2W	LWTTIHEII	0.813	WB	0.78	NS
	I9W	LQTTIHEIW	1.742	WB	0.42	NS
13	Original	LYCYEQFND	21.5	NB	0.72	NS
	D9F	LYCYEQFNF	0.129	SB	6.02	HS
	D9W	LYCYEQFNW	0.324	SB	3.89	WS
14	Original	QYNKPLCDL	0.742	WB	0.79	NS
	C7Y	QYNKPLYDL	0.139	SB	1.35	NS
	L9F	QYNKPLCDF	0.193	SB	1.62	NS
	L9W	QYNKPLCDW	0.388	SB	1.16	NS
15	Original	RCINCQKPL	37.667	NB	0.31	NS
	C2Y	RYINCQKPL	0.803	WB	1.29	NS
16	Original	RHLDKKQRF	0.718	WB	0.61	NS
	H2Y	RYLDKKQRF	0.013	SB	5.72	WS
	Q7Y	RHLDKKYRF	0.438	SB	1.17	NS
	H2W	RWLDKKQRF	0.094	SB	1.21	NS
17	Original	TDLYCYEQF	5.845	NB	0.75	NS
	D2Y	TYLYCYEQF	0.049	SB	13.74	HS
	D2W	TWLYCYEQF	0.299	SB	3.06	WS
18	Original	TTLEQQYNK	16.818	NB	0.33	NS
	T2Y	TYLEQQYNK	1.915	WB	1.21	NS
	K9F	TTLEQQYNF	0.57	WB	0.95	NS
	K9W	TTLEQQYNW	1.054	WB	0.72	NS
19	Original	VDIRTLEDL	16.436	NB	0.35	NS
	D2Y	VYIRTLEDL	0.114	SB	3.17	WS
	D2W	VWIRTLEDL	0.755	WB	0.73	NS
20	Original	VYGTTLEQQ	7.977	NB	0.74	NS
	Q9F	VYGTTLEQF	0.004	SB	6.61	HS
	Q9W	VYGTTLEQW	0.024	SB	4.54	WS

SB, strong binding; NB, no binding; WB, weak binding; NS, unstable; WS, weak stability; HS, strong stability.

Discussion

Polypeptides in the MHC peptide binding groove have been shown to mediate the recognition of T cells and other receptors by affecting the binding function of the complex. Peptides could regulate the movement of MHC itself, thereby prompting the recognition of the peptide-MHC complex by other receptors. Structural modeling of the peptide-MHC complex may reveal unknown driving factors for T cell activation, thereby contributing to the development of better and safer immunotherapy (29). In the present study, we found the intermolecular interactions of the polypeptide-HLA-A*2402 complex were maintained mainly by HB, followed by pi-stack. The binding affinity calculated by molecular docking also showed a significant negative correlation with the intermolecular force pi-stack and the pi-stack had a significant negative correlation with the binding affinity and binding free energy. The residues (C, D, Q, H, L, P, T) at position 2 on the polypeptide that did not form the intermolecular pi-stack force with HLA-A*2402 were replaced by Y and W, the residue (I) at position 4 was replaced by Y, the residues (T, C, Q) at position 7 were replaced by Y, the residues (C, D, K, Q, S, Y) at position 9 were replaced by F and W, and the predictive values of affinity and/or stability were improved when compared to the previous replacement.

Current studies have shown that the substitution of proline (Pro) for the third residue on the polypeptide could not only significantly enhance the ability of anti-tumor CTL to recognize wild-type epitopes (30), but also increase the affinity of pMHC and the stability of the complex (31). After analyzing the crystal structure of the MHC-peptide complex, the conformation of the modified antigen polypeptide was found to be like the wild type, and it interacted with the most conserved tyrosine residue Y159 in mammalian MHC-I molecules and maintained a stable bond (32). Changes in the identity of anchor residues may have significant effects, such as changing the conformation of the peptide-MHC complex, thereby affecting contact between the residues on the polypeptides and TCRs. Binding of the TCR-like recombinant antibody to the melanoma differentiation antigen gp100 T cell epitope G9-209 were completely dependent on the identity of the second single peptide anchor residue. In other words, the TCR-like antibody could only be modified with high affinity to HLA-A2 peptide G9-209-2M and then be recognized by specific complexes after contacting. It was suggested that the modification of anchor residues could significantly affect the conformation of the MHC peptide groove, which may have a profound impact on the interaction of TCR-pMHC molecules (33,34). Compared with non-antigenic peptides, antigenic peptides tend to bind to MHC-I molecules more stably, and results confirm that the unsuitable anchor residue at position 2 of the polypeptide is particularly prone to unstable interaction with MHC-I (13). The in vitro competitive binding ability after residue substitution at the above sites still requires further clarification in in vitro tests, and we will perform the Enzyme-Linked Immunosorbent spot (ELISpot) assay (35), immune repertoire (36,37) and peptide-MHC tetramer staining (38) to verify the prediction results, moreover, other factors that affect the affinity and stability of the polypeptides with HLA-A*2402 will require multidisciplinary, multidimensional analysis and discussion.

Conclusions

The generation and increase in the numbers of pi-stack interactions between antigen peptides and HLA-A*2402 may help improve the affinity and stability of the complex. The prediction of peptide-HLA intermolecular force and binding affinity by means of molecular docking is a supplement to the current commonly used prediction databases.

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