Crystal Structure of Phosphate Binding Protein Labeled with a Coumarin Fluorophore, a Probe for Inorganic Phosphate ²,‡ Miriam Hirshberg,* ,§ Kim Henrick, §,| Lesley Lloyd Haire, § Nishi Vasisht, § Martin Brune, ⊥ John E. T. Corrie, ⊥ and Martin R. Webb ⊥ DiVision of Protein Structure, National Institute for Medical Research, Mill Hill, London NW7 1AA, U.K., and DiVision of Physical Biochemistry, National Institute for Medical Research, Mill Hill, London NW7 1AA, U.K. ReceiVed February 23, 1998; ReVised Manuscript ReceiVed May 12, 1998 ABSTRACT: Crystal structures are presented for the A197C mutant of Escherichia coli phosphate binding protein (PBP) and the same mutant labeled at Cys197 with N-[2-(1-maleimidyl)ethyl]-7-(diethylamino)- coumarin-3-carboxamide (MDCC). Both proteins are complexed with inorganic phosphate. The latter molecule, MDCC-PBP, exhibits a large increase in fluorescence on binding inorganic phosphate. The resulting high-fluorescence state of the coumarin and the ability of this coumarin to monitor the conformational changes associated with inorganic phosphate binding are interpreted in terms of the specific interactions of MDCC with the protein. The structure helps to explain why this particular label gives a high-fluorescence state on binding inorganic phosphate, while several other related labels do not, and hence aids our general understanding of environmentally sensitive fluorescence probes on proteins. In Gram-negative bacteria, binding proteins located in the periplasm are vital components of the active transport system of a large variety of essential molecules, including carbo- hydrates, amino acids, and inorganic ions. Inorganic phos- phate (P i ) is bound by the phosphate binding protein (PBP), 1 which is a member of this class of proteins. PBP is produced under conditions of low P i concentration, binds P i in the periplasm, and transfers it to a membrane protein that transports it into the cytoplasm. Upon binding P i , PBP undergoes a large conformational change (1), which seems to be a common feature of proteins belonging to the binding protein family (2). The ability to monitor rapid changes of P i concentration in real time contributes to our understanding of biological processes such as systems involving ATP and GTP hydroly- sis. For this purpose, the A197C mutant of Escherichia coli PBP, labeled with a coumarin fluorophore [N-[2-(1-male- imidyl)ethyl]-7-(diethylamino)coumarin-3-carboxamide, MDCC] at Cys197, was developed as a fluorescent probe (MDCC-PBP) (3). Note that the maleimide moiety of MDCC becomes a succinimide as a consequence of its reaction with a thiol (Figure 1). The label at position 197 is on the edge of the P i binding site, close enough to monitor the conformational changes associated with P i binding without blocking the binding site itself. The observed increase in fluorescence intensity of MDCC-PBP upon binding P i (3, 4) presumably reflects the changes in the immediate environment of the coumarin as a consequence of the overall repositioning of the two domains of PBP. Its rapid response and high sensitivity have enabled this probe to be used in a number of different systems to measure P i release kinetics in real time (3, 5-8) The development of this probe has led us to investigate in detail the mechanism of P i binding to PBP. In the preceding paper (4), kinetic and spectroscopic measurements were used to study the P i binding and to characterize the resultant high-fluorescence state. In this paper, we describe the crystal structures of both the unlabeled A197C PBP‚P i and MDCC-PBP‚P i and consider the structural features that may cause the high-fluorescence state of the coumarin. The high-resolution structures of native PBP‚P i and the T141D mutant in both P i -bound and P i -free forms have been reported (1, 9). Comparison between these structures and the ones presented here allowed analysis of the effects of both the single mutation (A197C) and the labeling on PBP structure. Moreover, using the structure of P i -free T141D ² This work was funded by the Medical Research Council, U.K. ‡ The coordinates of the following structures have been deposited in the Brookhaven Protein Data Bank: A197C PBP‚Pi (entry 1a54) and MDCC-PBP‚Pi (entry 1a55). * To whom correspondence should be addressed. Telephone: (44) 181 3666, ext 2520. Fax: (44) 181 906 4477. E-mail: m-hirshb@ anika.nimr.mrc.ac.uk. § Division of Protein Structure, National Institute for Medical Research. | Current address: European Bioinformatics Institute, Hinxton, Cambridge CB10 1SD, UK ⊥ Division of Physical Biochemistry, National Institute for Medical Research. 1 Abbreviations: PBP, phosphate binding protein; MDCC, N-[2-(1- maleimidyl)ethyl]-7-(diethylamino)coumarin-3-carboxamide; MDCC- PBP, A197C mutant of PBP labeled with MDCC at the cysteine; IDCC, N-[2-(iodoacetamido)ethyl]-7-(diethylamino)coumarin-3-carboxa- mide. FIGURE 1: Structure of the MDCC-thiol adduct. 10381 Biochemistry 1998, 37, 10381-10385 S0006-2960(98)00428-0 CCC: $15.00 © 1998 American Chemical Society Published on Web 07/01/1998