The structure of the liganded form of phosphate-binding protein, an essential component of the Pst inorganic phosphate active transport system of E. coli has been determined and refined at 1.7 A resolution to an R-factor of 14.7% for 32,222 reflections. The initial model was derived by fitting the amino acid sequence to a 2.5 A resolution electron-density map computed with isomorphous replacement phases from a single iodo derivative with anomalous dispersion data. The mean figure of merit is 0.69 for 11,477 reflections. The model consists of all 321 amino acid residues (2,439 non-hydrogen atoms), the phosphate substrate and 259 ordered water molecules.
The molecule is ellipsoid with overall dimensions of 35 A $\times$ 40 A $\times$ 70 A. Phosphate-binding protein structurally resembles six other periplasmic binding proteins (specific for sc L-arabinose, sc D-galactose/ sc D-glucose, leucine/isoleucine/valine, leucine, sulfate and maltose) solved in our laboratory. The molecule consists of two domains, each with a central $\beta$-sheet flanked by $\alpha$-helices. The domains are connected by a hinge which is composed of three strands and one helix. The substrate is bound in the cleft formed between the domains. The general folding pattern is parallel $\alpha$/$\beta$ with the exception of one antiparallel strand in each $\beta$-sheet as a result of the crossover between the domains.
The bound phosphate anion is inaccessible to the bulk solvent. It interacts with the protein through twelve hydrogen bonds.
Molecular dynamics simulations using free energy perturbation techniques reproduce the experimentally observed specificity of phosphate-binding protein. A decreased affinity is almost entirely due to differences in electrostatic interactions.
Electrostatic field calculations based on the finite difference method compute a strong field gradient for the binding site in the absence of the substrate, caused by the proximity of several ionized side chains as well as a number of peptide amides and polar side chains. The gradient is complementary to the net dipole moment of the bound HPO$\sb4\sp{=}$. The electrostatic potential at the binding site is positive averaging about 100 kT/e despite the uncompensated charge of Asp56. (Abstract shortened with permission of author.)
| Identifer | oai:union.ndltd.org:RICE/oai:scholarship.rice.edu:1911/16367 |
| Date | January 1990 |
| Creators | Luecke, Hartmut |
| Contributors | Quiocho, Florante A. |
| Source Sets | Rice University |
| Language | English |
| Detected Language | English |
| Type | Thesis, Text |
| Format | 163 p., application/pdf |
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