Margaret Vandonselaar

The 1·6 Å structure of histidine-containing phosphotransfer protein HPr from Streptococcus faecalis

The histidine-containing phosphocarrier protein (HPr) is a central component of the phosphoenolpyruvate: sugar phosphotransferase system (PTS) that transports carbohydrates across the cell membrane of bacteria. The three-dimensional structure of Gram-positive Streptococcus faecalis HPr has been determined using the method of multiple isomorphous replacement. The R factor for all data is 0.156 for S. faecalis HPr at 1.6 A resolution with very good geometry. The overall folding topology of HPr is a classical open-faced beta-sandwich, consisting of four antiparallel beta-strands and three alpha-helices. Remarkable disallowed Ramachandran torsion angles of Ala16 at the active center, revealed by the X-ray structure of S. faecalis HPr, demonstrate a unique example of torsion-angle strain that is likely involved directly in protein function. A brief report concerning the torsion-angle strain has been presented recently. A newly-determined pH 7.0 structure is shown to have the same open conformation of the active center and the same torsion-angle strain at Ala16, suggesting that pH is not responsible for the structural observations. The current structure suggests a role for residues 12 and 51 in HPrs function, since they are involved in the active center through direct and indirect hydrogen-bonding interactions with the imidazole ring of His15. It is found that Ser46, the regulatory site in HPr from Gram-positive bacteria, N-caps the minor alpha-B helix and is also involved in the Asn43-Ser46 beta-turn. This finding, in conjunction with the proposed routes of communication between the regulatory site Ser46 and the active center in S. faecalis HPr, provides new insight into the understanding of how Ser46 might function. The putative involvement of the C-terminal alpha-carboxyl group and the related Gly67-Glu70 reverse beta-turn with respect to the function of HPr are described.

Crystallization of the calcium-activated phosphoenolpyruvate carboxykinase from Escherichia coli K12.

Single crystals of phosphoenolpyruvate carboxykinase from Escherichia coli K12 have been grown in the orthorhombic crystal system. Single crystals developed to a maximum size of 0.25 mm x 0.25 mm x 1.5 mm by the technique of washing and reseeding. The space group is P2(1)2(1)2(1), with a = 77.24 A, b = 89.18 A, c = 93.24 A and Z = 4; there is one enzyme molecule per crystallographic asymmetric unit and the solvent content is estimated to be 59%. The crystals diffract to at least 2.8 A d spacings and decompose in the X-ray beam after approximately two days of exposure.

Preliminary crystallographic data for a monoclonal Fab fragment specific for HPr of the phosphoenolpyruvate: Sugar phosphotransferase system of Escherichia coli

Crystals for Fab fragments from a monoclonal antibody to HPr of the phosphoenopyruvate:sugar phosphotransferase system of Escherichia coli have been obtained from 14% polyethylene glycol 6000, 5 mM-Tris X HCl, 50 mM-sodium phosphate and 0.2 M-sodium chloride at pH 8.0. The space group is P2(1) with a = 110.85 A, b = 66.18 A, c = 67.21 A, beta = 113.0 degrees and Z = 4. The crystals exhibit the forms [100], [011] and [011] and the solvent content is 47%.

Protein crystal growth aboard the U.S. space shuttle flights STS-31 and STS-32

The first microgravity protein crystal growth experiments were performed on Spacelab I by Littke and John. These experiments indicated that the space grown crystals, which were obtained using a liquid-liquid diffusion system, were larger than crystals obtained by the same experimental system on earth. Subsequent experiments were performed by other investigators on a series of space shuttle missions from 1985 through 1990. The results from two of these shuttle flights (STS-26 and STS-29) have been described previously. The results from these missions indicated that the microgravity grown crystals for a number of different proteins were larger, displayed more uniform morphologies, and yielded diffraction data to significantly higher resolutions than the best crystals of these proteins grown on earth. This paper presents the results obtained from shuttle flight STS-32 (flown in January, 1990) and preliminary results from the most recent shuttle flight, STS-31 (flown in April, 1990).