Table 1.

Recognition by MK16 Is Affected by Single Amino Substitutions and Truncations of the MBP 85–99 Peptide

	Residue no. and position in the binding pocket
	85	86	87	88	89	90	91	92	93	94	95	96	97	98	99
		P-3	P-2	P-1	P1	P2	P3	P4	P5	P6	P7	P8	P9	P10	P11
Peptide	Alanine analogs																			IC50 MK16	IC50 DRB1*1501
																nM	nM
(85–89)	E	N	P	V	V	H	F	F	K	N	I	V	T	P	R	6	5
85 (E→A)	A	N	P	V	V	H	F	F	K	N	I	V	T	P	R	4	7
86 (N→A)	E	A	P	V	V	H	F	F	K	N	I	V	T	P	R	2	7
87 (P→A)	E	N	A	V	V	H	F	F	K	N	I	V	T	P	R	4	10
88 (V→A)	E	N	P	A	V	H	F	F	K	N	I	V	T	P	R	4	10
89 (V→A)	E	N	P	V	A	H	F	F	K	N	I	V	T	P	R	4	50
90 (H→A)	E	N	P	V	V	A	F	F	K	N	I	V	T	P	R	13	10
91 (F→A)	E	N	P	V	V	H	A	F	K	N	I	V	T	P	R	9	10
92 (F→A)	E	N	P	V	V	H	F	A	K	N	I	V	T	P	R	217	199
93 (K→A)	E	N	P	V	V	H	F	F	A	N	I	V	T	P	R	18	4
94 (N→A)	E	N	P	V	V	H	F	F	K	A	I	V	T	P	R	19	4
95 (I→A)	E	N	P	V	V	H	F	F	K	N	A	V	T	P	R	10	4
96 (V→A)	E	N	P	V	V	H	F	F	K	N	I	A	T	P	R	131	4
97 (T→A)	E	N	P	V	V	H	F	F	K	N	I	V	A	P	R	5	4
98 (P→A)	E	N	P	V	V	H	F	F	K	N	I	V	T	A	R	>1,000	5
99 (R→A)	E	N	P	V	V	H	F	F	K	N	I	V	T	P	A	>1,000	5
NH2- or COOH-terminal truncations
(85–99)	–	N	P	V	V	H	F	F	K	N	I	V	T	P	R	2	3
(87–99)	–	–	P	V	V	H	F	F	K	N	I	V	T	P	R	2	5
(88–99)	–	–	–	V	V	H	F	F	K	N	I	V	T	P	R	4	30
(89–99)	–	–	–	–	V	H	F	F	K	N	I	V	T	P	R	9	253
(90–99)	–	–	–	–	–	H	F	F	K	N	I	V	T	P	R	>1,000	>1,000
(91–99)	–	–	–	–	–	–	F	F	K	N	I	V	T	P	R	>1,000	>1,000
(85–98)	E	N	P	V	V	H	F	F	K	N	I	V	T	P	–	>1,000	4
(85–97)	E	N	P	V	V	H	F	F	K	N	I	V	T	–	–	>1,000	15
(85–96)	E	N	P	V	V	H	F	F	K	N	I	V	–	–	–	>1,000	20
Single amino acid substitutions
89 (V→D)	E	N	P	V	D	H	F	F	K	N	I	V	T	P	R	214	281
92 (F→Y)	E	N	P	V	V	H	F	Y	K	N	I	V	T	P	R	6	12
92 (F→D)	E	N	P	V	V	H	F	D	K	N	I	V	T	P	R	>1,000	>1,000

MK16 Fab-phages were mixed with various concentrations of purified DRB1*1501 molecules loaded with peptides containing the indicated single amino acid substitutions or truncations, followed by incubation in microtiter wells coated with DRB1*1501–MBP 85–99 peptide complexes. Bound MK16 Fab-phages were detected with biotin-labeled rabbit anti–Fd bacteriophage by ELISA. For peptide binding and specificity of purified DRB1*1501 molecules isolated from S2/DRB1*1501 cells, a constant amount of DRB1*1501 molecules was incubated in the presence of a fixed concentration of biotin-labeled MBP 85–99 peptide and increasing amounts of competitor peptide. The concentration of competitor complex needed to result in IC₅₀ was determined. Binding values for residues critical for binding are in bold type. Each peptide was analyzed in three independent experiments, and results are represented as mean values.