The bacterial melibiose permease (MelB) belongs to the glycoside-pentoside-hexuronide:cation symporter family (GPH), a part of the major facilitator superfamily (MFS). Structural information regarding GPH transporters and other Na+-coupled permeases within MFS has been lacking, although a wealth of biochemical and biophysical data are available. Here we present the 3D crystal structures of Salmonella typhimurium MelBSt in two conformations, representing an outward partially occluded and an outward inactive state of MelBSt. MelB adopts a typical MFS fold, and contains a previously unidentified cation-binding motif. Three conserved acidic residues form a pyramidal-shaped cation-binding site for Na+, Li+, or H+, which is in close proximity to the sugar-binding site. Both co-substrate-binding sites are mainly contributed by the residues from the N-terminal domain. These two structures and the functional data presented here provide mechanistic insights into Na+/melibiose symport. We also postulate a structural foundation for the conformational cycling necessary for transport catalyzed by MFS permeases in general.

The three-dimensional crystal structure of an arginine kinase transition-state analogue complex has been re®ned at 1.2 A Ê resolution, with an overall R factor of 12.3%. The current model provides a unique opportunity to analyze the structure of a bimolecular (phosphagen kinase) enzyme in its transition state. This atomic resolution structure con®rms in-line transfer of the phosphoryl group and the catalytic importance of the precise alignment of the substrates. The structure is consistent with a concerted proton transfer that has been proposed for an unrelated kinase. Re®nement of anisotropic temperature factors and translation±libration±screw (TLS) analyses led to the identi®cation of four rigid groups and their prevalent modes of motion in the transition state. The relative magnitudes of the mobility of rigid groups are consistent with their proposed roles in catalysis.

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Arginine kinase (AK) is a member of the guanidino kinase family that plays an important role in buffering ATP concentration in cells with high and fluctuating energy demands. The AK specifically catalyzes the reversible phosphoryl transfer between ATP and arginine. We have determined the crystal structure of AK from the horseshoe crab (Limulus polyphemus) in its open (substrate-free) form. The final model has been refined at 2.35 Å with a final R of 22.3% (R free ‫ס‬ 23.7%). The structure of the open form is compared to the previously determined structure of the transition state analog complex in the closed form. Classically, the protein would be considered two domain, but dynamic domain (DynDom) analysis shows that most of the differences between the two structures can be considered as the motion between four rigid groups of nonsequential residues. ATP binds near a cluster of positively charged residues of a fixed dynamic domain. The other three dynamic domains close the active site with separate hinge rotations relative to the fixed domain. Several residues of key importance for the induced motion are conserved within the phosphagen kinase family, including creatine kinase. Substantial conformational changes are induced in different parts of the enzyme as intimate interactions are formed with both substrates. Thus, although induced fit occurs in a number of phosphoryl transfer enzymes, the conformational changes in phosphagen kinases appear to be more complicated than in prior examples.

Arginine kinase is a member of the phosphagen kinase family that includes creatine kinase and likely shares a common reaction mechanism in catalyzing the buffering of cellular ATP energy levels. Abstraction of a proton from the substrate guanidinium by a catalytic base has long been thought to be an early mechanistic step. The structure of arginine kinase as a transition state analog complex (Zhou, G., Somasundaram, T., Blanc, E., Parthasarathy, G., Ellington, W. R., and Chapman, M. S. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 8449 -8454) showed that Glu-225 and Glu-314 were the only potential catalytic residues contacting the phosphorylated nitrogen. In the present study, these residues were changed to Asp, Gln, and Val or Ala in several single and multisite mutant enzymes. These mutations had little impact on the substrate binding constants. The effect upon activity varied with reductions in k cat between 3000-fold and less than 2-fold. The retention of significant activity in some mutants contrasts with published studies of homologues and suggests that acid-base catalysis by these residues may enhance the rate but is not absolutely essential. Crystal structures of mutant enzymes E314D at 1.9 Å and E225Q at 2.8 Å resolution showed that the precise alignment of substrates is subtly distorted. Thus, pre-

The melibiose permease of Escherichia coli (MelB) catalyzes the coupled stoichiometric symport of a galactoside with a cation (either Na ؉ , Li ؉ , or H ؉ ), using free energy from the downhill translocation of one cosubstrate to catalyze the accumulation of the other. Here, we present a 3D structure model of MelB threaded through a crystal structure of the lactose permease of E. coli (LacY), manually adjusted, and energetically minimized. The model contains 442 consecutive residues (Ϸ94% of the polypeptide), including all 12 transmembrane helices and connecting loops, with no steric clashes and superimposes well with the template structure. The electrostatic surface potential calculated from the model is typical for a membrane protein and exhibits a characteristic ring of positive charges around the periphery of the cytoplasmic side. The 3D model indicates that MelB consists of two pseudosymmetrical 6-helix bundles lining an internal hydrophilic cavity, which faces the cytoplasmic side of the membrane. Both sugar and cation binding sites are proposed to lie within the internal cavity. The model is consistent with numerous previous mutational, biochemical/biophysical characterizations as well as low-resolution structural data. Thus, an alternating access mechanism with sequential binding is discussed. The proposed overall fold of MelB is different from the available crystal structures of other Na ؉ -coupled transporters, suggesting a distinctive fold for Na ؉ symporters.bioenergetics ͉ ligand binding ͉ MelB ͉ protein threading ͉ sugar/cation symporter M elibiose permease of Escherichia coli (MelB), encoded by the melB gene in the mel operon (1), is a well-studied representative of the glycoside-pentoside-hexuronide/cation symporter family of membrane transporters (2). MelB utilizes free energy released from the energetically downhill movement of a cation (Na ϩ , Li ϩ , or H ϩ ), in response to an electrochemical cation gradient, to drive the uphill stoichiometric accumulation of a galactopyranoside (3-5). The type of the cotransported cation depends on the stereostructure of the transported sugar (6). ␣-Galactopyranosides (melibiose, raffinose, and p-nitrophenyl-␣-galactoside) are cotransported with Na ϩ , H ϩ , or Li ϩ , whereas ␤-galactopyranosides (lactose, methyl-1-␤-D-galactopyranoside, and p-nitrophenyl-␤-galactoside) are cotransported with Na ϩ or Li ϩ but not H ϩ (6). In the absence of an electrochemical cation gradient, MelB catalyzes the reverse reaction, using free energy from the downhill translocation of the sugar to drive the stoichiometric transport of the coupled cation in either direction across the membrane (5,7,8). Similar features of transport exist in the lactose permease of E. coli (LacY) (9, 10), the best-studied representative of the major facilitator superfamily (MFS) of membrane transporters (2). Both MelB and LacY transport D-galactopyranosides, with similar substrate specificity; however, sugar transport in LacY is coupled solely with H ϩ (9, 10). MelB consists of 473 aa with Ϸ65% apo...

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