Extending the diffraction limit of protein crystals: The example of glutamine synthetase from Salmonella typhimurium in the presence of its cofactor ATP

Abstract

Glutamine synthetase (GS) plays a central role in cellular nitrogen metabolism in bacteria, catalyzing the ATPdependent condensation of ammonia with glutamate to yield glutamine in the presence of divalent cations. GS is regulated by product feedback, by oxidative modification, and by adenylylation (Rhee et al., 1989). The 3.5-A structure of completely unadenylylated GS was determined by Almassy et al. (1986) and refined by Yamashita et al. (1989). To improve the atomic model, and our understanding of the regulation and reaction mechanism, it has been necessary to grow GS crystals that diffract to higher resolution. We find that the cofactor ATP affects the size, growth rate, and diffraction quality of GS crystals.Two crystal forms grown in different concentrations of ATP or the reaction products ADP and Pi are found to have distinct morphologies, stabilities, space groups, and resolution limits. Form XVI crystals, grown at high ATP concentration (greater than 500 pM), grow to greater than 1 mm, belong to space group C222, and have unit-cell constants a = 235.1 A , b = 138.7 A , and c = 205.6 A.They diffract to no better than 3.2 A resolution and have a short lifetime in the X-ray beam. These crystals tend to redissolve.Form XIV crystals are grown at low ATP concentrations (less than 200 pM). They are routinely 0.8 X 0.6 X 0.2 mm and belong to space group C2, with unit-cell dimensions a = 235.5 A , b = 134.5 A, c = 200.1 A , and / 3 = 102.8'. They diffract to 2.4 A resolution, are suitable for data collection, and are stable for at least 6 months. Both Form XIV and XVI crystals have been observed earlier (Heidner et al., 1978;Janson et al., 1984), and Form XIV crystals have been used to determine the GS structure at 3.5 A resolution. However, before we determined the importance of ATP, crystals grew irregularly, were rarely large enough for X-ray diffraction studies, and had limited resolution. The present study reveals that the ATP concentration is a crucial variable in obtaining large, better diffracting crystals.The two differences in the growth conditions between crystal Forms XIV and XVI are the concentrations of ATP and of spermine tetrahydrochloride. The ATP:GS monomer ratio is 0.5 in the best Form XIV crystals, and it is equal to or greater than 1 in Form XVI crystals.Two lines of evidence suggest that high ATP concentration makes GS more soluble, accounting for the redissolving of Form XVI crystals. The first is that increased concentrations of positively charged spermine tetrahydrochloride stabilize the growth of GS containing high ATP. This is presumably because of charge compensation. The other line of evidence is that addition of 2 mM ATP to crystallization drops dissolves Form XIV crystals. High ATP may change the packing of GS moIecules, either because of an additional ATP binding site on the GS monomer, or because of the added negative charges of ATP. Whether ATP actually alters the packing of GS molecules we will learn only when we determine the structure of Form XVI crystals.It may be significa...

Structural basis for the helical filament formation of Escherichia coli glutamine synthetase

“…Next, we focused on the catalytic pocket of EcGS where ATP and glutamate are coupled via Mg 2+ coordination to generate the intermediate product γglutamylphosphate. 1,22 As expected, no density correlating to ATP or glutamate could be detected in our cryo-EM structure (Figure 5b), implying that GS filaments represent polymerization of inactive GS dodecamers (apo form). Notably, we did detect an unknown density surrounded by charged residues, including Glu130, His270, and Glu358.…”

A model for oxidative modification of glutamine synthetase, based on crystal structures of mutant H269N and the oxidized enzyme

“…Ginsburg and co-workers termed the catalytically active GS "taut" in contrast to the inactive relaxed GS from which metal ions have been removed by EDTA (Ginsburg, 1972). Consistent with this hypothesis of the importance of electrostatic forces to the stability of GS, addition of more than 2 mM ATP or ADP to crystallization drops dissolves crystals, perhaps due to the added negative charges (Liaw et al, 1993).…”

“…Crystal Growth. Crystallization conditions of H269N from E. coli are similar to those of native GS from S. typhimurium (Liaw et al, 1993). Crystals are grown by the hanging drop method of vapor diffusion.…”

Nitrogen Assimilation in Escherichia coli: Putting Molecular Data into a Systems Perspective

“…A cylindrical active site is formed by six antiparallel ␤-strands of one subunit and two strands of the neighboring subunit and holds two divalent cations (Mn 2ϩ ions) as cofactors (36) necessary for catalysis (37). The substrate binding sites of glutamate, ATP, and ammonium are located within this active site (36,(38)(39)(40)(41). GS has 12 active sites that may well act cooperatively (see below).…”