SHORT HYDROGEN BONDS IN A PROTEIN RECEPTOR-PHOSPHATE COMPLEX: EVIDENCE FROM STRUCTURES REFINED AT 1 RESOLUTION. Zhongmin Wang*, Hartmut Luecke, and Florante A. Quiocho*,. *Structural and Computational Biology and Molecular Biophysics Program, Howard Hughes Medical Institute and Department of Biochemistry, Baylor College of Medicine, Houston, Texas 77030 and Stanford Synchrotron Radiation Laboratory, Stanford University, Stanford, CA 94309
The protein receptor (a phosphate-binding protein or PBP with a mass of 33,000 daltons) serves as an initial and extremely specific component of bacterial active transport. Fully consistent with the stringent specificity of PBP, the initial 1.7 structure of the complex shows that the completely dehydrated and sequestered phosphate forms 12 hydrogen bonds (11 with donor groups and 1 with an acceptor group) in addition to one salt link with a guanidinium group. The distance of the hydrogen bond between the donor phosphate O4 oxygen and the acceptor group (an oxygen of carboxylate of Asp 56) is 2.45 . This short hydrogen bond has also been observed in the 1.9 structure of a fully active mutant PBP in which the donor group Thr141 was replaced by an acceptor Asp residue and in several high resolution structures of other PBP mutants. Moreover, the Asp141 substitution further introduced another short hydrogen bond (2.50 ) between the phosphate O2 oxygen and an Asp141. In order to cement these findings of short hydrogen bonds, as well as obtain an atomic structure, excellent synchrotron data for the wild-type and the Asp141 mutant PBP were collected to resolutions of 0.98 and 1.00 , respectively. Using SHELXL-93, full matrix refinement of both structures with hydrogen atoms and anisotropic B-factor for non-hydrogen atoms against the ultra high resolution data confirmed the existence of the short hydrogen bonds. These short hydrogen bonds in the PBP-phosphate complexes could be classified as low barrier hydrogen bonds with energies ranging from 12 to 24 kcal/mole 1,2,3. Nevertheless, the Kd values of 1 - 10 (M for the complexes do not reflect these high energy hydrogen bonds. We thank Dr. George M. Sheldrick for providing the program SHELXL and helpful advice.
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