Identification by NMR of the binding surface for the histidine-containing phosphocarrier protein HPr on the N-terminal domain of enzyme I of the Escherichia coli phosphotransferase system

被引:206
作者
Garrett, DS
Seok, YJ
Peterkofsky, A
Clore, GM
Gronenborn, AM
机构
[1] NIDDKD,CHEM PHYS LAB,NIH,BETHESDA,MD 20892
[2] NHLBI,BIOCHEM GENET LAB,NIH,BETHESDA,MD 20892
关键词
D O I
10.1021/bi970221q
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The interaction between the similar to 30 kDa N-terminal domain of enzyme I (EIN) and the similar to 9.5 kDa histidine-containing phosphocarrier protein HPr of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system has been investigated by heteronuclear magnetic resonance spectroscopy. The complex is in fast exchange, permitting us to follow the chemical shift changes of the backbone NH and N-15 resonances of EIN upon complex; formation by recording a series of H-1-N-15 correlation spectra of uniformly N-15-labeled EIN in the presence of increasing amounts of HPr at natural isotopic abundance. The equilibrium association constant derived from analysis of the titration data is similar to 1.5 x 10(5) M-1, and the lower limit for the dissociation rate constant is 1100 s(-1). By mapping the backbone chemical shift perturbations on the three-dimensional solution structure of EIN [Garrett, D. S., Seek, Y.-J., Liao, D.-I., Peterkofsky, A., Gronenborn, A. M., & Clore, G. M. (1997) Biochemistry 36, 2517-2530], we have identified the binding surface of EIN in contact with HPr. This surface is primarily located in the ct domain and involves helices H1, H2, and H4, as well as the hinge region connecting helices H2 and H2'. The data also indicate that the active site His 15 of HPr must approach the active site His 189 of EIN along the shallow depression at the interface of the cr and alpha/beta domains. Interestingly, both the backbone and side chain resonances (assigned from a long-range H-1-N-15 correlation spectrum) of His 189, which is located at the N-terminus of helix H6 in he alpha/beta domain, are only minimally perturbed upon complexation, indicating that His 189 (in the absence of phosphorylation) does not undergo any significant conformational change or change in pK(a) value upon HPr binding. On the basis of results of this study, as well as a previous study which delineated the interaction surface for EI on HPr [van Nuland, N. A. J., Boelens, R., Scheek, R. M., & Robillard, G. T. (1995) J. Mol. Biol. 246, 180-193], a model for the EIN/HPr complex is proposed in which helix 1 (residues 16-27) and the helical loop (residues 49-53) of HPr slip between the two pairs of helices constituting the ct domain of EIN. In addition, we suggest a functional role for the kink between helices H2 and H2' of EIN, providing a flexible joint for this interaction to take place.
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页码:4393 / 4398
页数:6
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共 34 条
[11]   The solution structure of HIV-1 Nef reveals an unexpected fold and permits delineation of the binding surface for the SH3 domain of Hck tyrosine protein kinase [J].
Grzesiek, S ;
Bax, A ;
Clore, GM ;
Gronenborn, AM ;
Hu, JS ;
Kaufman, J ;
Palmer, I ;
Stahl, SJ ;
Wingfield, PT .
NATURE STRUCTURAL BIOLOGY, 1996, 3 (04) :340-345
[12]   THE IMPORTANCE OF NOT SATURATING H2O IN PROTEIN NMR - APPLICATION TO SENSITIVITY ENHANCEMENT AND NOE MEASUREMENTS [J].
GRZESIEK, S ;
BAX, A .
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 1993, 115 (26) :12593-12594
[13]   REEXAMINATION OF THE SECONDARY AND TERTIARY STRUCTURE OF HISTIDINE-CONTAINING PROTEIN FROM ESCHERICHIA-COLI BY HOMONUCLEAR AND HETERONUCLEAR NMR-SPECTROSCOPY [J].
HAMMEN, PK ;
WAYGOOD, EB ;
KLEVIT, RE .
BIOCHEMISTRY, 1991, 30 (51) :11842-11850
[14]   UNRAVELING A BACTERIAL HEXOSE-TRANSPORT PATHWAY [J].
HERZBERG, O ;
KLEVIT, R .
CURRENT OPINION IN STRUCTURAL BIOLOGY, 1994, 4 (06) :814-822
[15]   STRUCTURE OF THE HISTIDINE-CONTAINING PHOSPHOCARRIER PROTEIN HPR FROM BACILLUS-SUBTILIS AT 2.0-A RESOLUTION [J].
HERZBERG, O ;
REDDY, P ;
SUTRINA, S ;
SAIER, MH ;
REIZER, J ;
KAPADIA, G .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1992, 89 (06) :2499-2503
[16]  
JIA ZC, 1993, J BIOL CHEM, V268, P22490
[17]   GRAPHICS MODEL-BUILDING AND REFINEMENT SYSTEM FOR MACROMOLECULES [J].
JONES, TA .
JOURNAL OF APPLIED CRYSTALLOGRAPHY, 1978, 11 (AUG) :268-272
[18]   TWO-DIMENSIONAL H-1-NMR STUDIES OF HISTIDINE-CONTAINING PROTEIN FROM ESCHERICHIA-COLI .3. SECONDARY AND TERTIARY STRUCTURE AS DETERMINED BY NMR [J].
KLEVIT, RE ;
WAYGOOD, EB .
BIOCHEMISTRY, 1986, 25 (23) :7774-7781
[19]   STRUCTURE OF THE IIA-DOMAIN OF THE GLUCOSE PERMEASE OF BACILLUS-SUBTILIS AT 2.2-A RESOLUTION [J].
LIAO, DI ;
KAPADIA, G ;
REDDY, P ;
SAIER, MH ;
REIZER, J ;
HERZBERG, O .
BIOCHEMISTRY, 1991, 30 (40) :9583-9594
[20]   The first step in sugar transport: Crystal structure of the amino terminal domain of enzyme I of the E-coli PEP: Sugar phosphotransferase system and a model of the phosphotransfer complex with HPr [J].
Liao, DI ;
Silverton, E ;
Seok, YJ ;
Lee, BR ;
Peterkofsky, A ;
Davies, DR .
STRUCTURE, 1996, 4 (07) :861-872