Inhibitory mechanism of peptides with a repeating hydrophobic and hydrophilic residue pattern on interleukin-10

Figures & data

Table 1. The sequences of P1, P2 and peptides modified based on P1 or P2.

Name	Sequence	Net charge	MW (Da)	Modification description
P1	FFKKF FKKFF KKFFK K-OH	+8	2220.84	—
P1.1	FFKKF FKKFF KKFF-OH	+6	1964.49	C-ter truncate (2 amino acids)
P1.2	FFKKF FKKFF KK-OH	+6	1670.14	C-ter truncate (4 amino acids)
P1.3	D-FFKKF FKKFF KKFFK K-OH	+8	2220.84	D-amino acid substitution
P1.4	FRKKF RKKFF KKFFK K-OH	+8	2238.82	Sequence pattern, (FxKK)2(FFKK)2^a^a,b
P1.5	FRKKF RKKFR KKFRK K-OH	+12	2256.85	Sequence pattern, (FxKK)4
P1.6	FFKAF FKAFF KAFFK A-OH	+4	1992.45	C-ter hydrophobic substitution and sequence pattern (FFKy)4^b^a,b
P2	FFRRF FRRFF RRFFR R-OH	+8	2444.95	—
P2.1	EFRRE FRREF RREFR R-OH	0	2372.70	N-ter hydrophilic substitution and helical structure stabilization by E-R bridges
P2.2	WYRRF YRRAH RRAHR R-OH	+8	2343.72	N-ter hydrophobicity decreased & C-ter helical structure stabilization by AH
P2.3	WYHHF YHHAH RRAHR R-OH	+4	2267.53	N-ter hydrophobicity decreased and helical structure stabilization by AH, HH and WH bridge
P3 (negative control)	GTELP SPPSV WFEAE F-OH	−3	1792.99	—

^a,b x and y represent hydrophilic and hydrophobic amino acids, respectively.

Figure 1. Effect of different TFE concentrations on the conformation of P1 and P2. Far-UV CD spectra of P1 (A) and P2 (B). Representative data are shown from 3 independent experiments performed in triplicate.

Figure 2. Interactions between P1/P2/P3 and IL-10R1 using Surface Plasmon Resonance Spectroscopy. Sensorgrams of the surface binding affinities of peptide P1 (A), P2 (B) and P3 (C) at different concentrations in relation with time, and (D) Binding affinity measurement of P1, P2 and P3 with IL-10R1.

Table 2. Hydrogen bonds between P1 and IL-10R1 complex [side chain NH (H_N), hydroxyl hydrogen (H_O); backbone amide nitrogen (H), backbone carbonyl oxygen (O), carboxylate oxygen (O_X) and hydroxyl oxygen (or amide-connected oxygen on side chain, O_H).

P1	IL-10R1	Bond length (Å)
Lys7: HN,1	Ser137: OH	1.94
Lys7: HN,2	Asp133: Ox	1.80
Lys8: HN,1	Ser137: O	2.36
Lys8: HN,2	Ser140: OH	2.23
Lys12: H	Asp99: OX,1	1.95
Lys12: HN,1	Arg95: O	2.13
Lys12: HN,2	Glu100: OX	1.80
Lys16: HN,1	Asp99: OX,2	1.75
Lys16: HN,2	Ser191: OH	2.05

Table 3. The dissociation free energy (ΔG⁰_D,solv, in kcal/mol) of IL-10R with IL-10, P1 or P2 (the full calculation results were listed in S6 Table).

		IL-10/IL-10R1	P1/IL-10R1	P2/IL-10R1
ΔG^0D,solv	PB	−39.46	−46.53	−62.39
	GB	−28.71	−40.75	−66.39

Figure 3. The effects of truncated and mutated P1 peptides on IL-10 level in U937 cells stimulated by LPS. Supernatants were measured for the presence of IL-10 by ELISA. The concentration of LPS is 4 × 10⁻³ µM: 3 × 10⁵ human U937 were either left unstimulated (UN) or stimulated with LPS, LPS + 0.1 µM of anti-IL10R, LPS + 0.3 µM of aIL10, LPS + 0.1 µM of aIL10R, L+P1, P1.1, P1.2, P1.3, P1.4, P1.5, P2 and P3 at 4.50 µM overnight, respectively. P values were calculated using 2 tailed Student's t-test.

Figure 4. The effects of mutated P2 peptides on IL-10 secretion in PBMCs stimulated by LPS. Supernatants were measured for the presence of IL-10 by ELISA. The concentration of LPS is 4 × 10⁻³ µM (100 ng): 1 × 10⁶ human U937 were either left unstimulated (UN) or stimulated with LPS, LPS + 0.3 µM of anti-IL10, LPS+0.1 µM of anti-IL10 receptor (aIL10R), LPS + P1, P2, P2.1, P2.2, P2.3 and P3 at 4.50 µM overnight, respectively. P values were calculated using 2 tailed Student's t-test.