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Alanine scanning mutagenesis of the charged amino acids of melanoma growth stimulating activity (MGSA) was used to identify specific residues that are involved in binding to the human erythrocyte Duffy antigen/chemokine receptor (DARC) and to the type B interleukin-8 receptor (IL-8RB) on neutrophils. Receptor binding and biological studies with the alanine scan mutants of MGSA demonstrate that MGSA binds to DARC and the IL-8RB through distinct binding regions. One of the MGSA mutants, E6A, binds to human erythrocytes and is able to inhibit malaria invasion as efficiently as wild type MGSA but has a severely reduced ability to bind to or signal through the IL-8RB. Mutant chemokines like E6A could prove to be useful therapeutically for the design of receptor blocking drugs that inhibit erythrocyte invasion by Plasmodium vivax malaria.

The solution structure of melanoma growth stimulating activity (MGSA), a dimeric chemokine consisting of 73 residues per monomer, has been determined using two-dimensional homonuclear and three-dimensional heteronuclear NMR spectroscopy. Structure calculations were carried out using a hybrid distance geometry-simulated annealing approach with the programs DGII and X-PLOR. The structure is based on a total of 2362 experimental restraints, comprising 2150 NOE-derived distance restraints (2076 unambiguous intrasubunit restraints, 60 unambiguous intersubunit restraints, and 14 ambiguous restraints with potential contributions from both intra- and intersubunit NOEs), 84 distance restraints for 42 backbone hydrogen bonds, and 128 torsion angle restraints. The ambiguous distance restraints were treated using a target function which accounts for both intra- and intermolecular contributions to the NOE intensity. A total of 25 structures were calculated, with the backbone (N, C alpha, C) atomic r.m.s. distribution about the mean coordinates for residues 8 to 69 being 0.44(+/- 0.10) A for the dimer and 0.34(+/- 0.07) A for the individual monomers. The N- and C-terminal residues (1 to 7 and 70 to 73, respectively) are disordered. The overall structure of the MGSA dimer is similar to that reported previously for the NMR and X-ray structures of interleukin-8 (IL-8), and consists of a six-stranded antiparallel beta-sheet packed against two C-terminal antiparallel alpha-helices. A best fit superposition of the NMR structure of MGSA on the X-ray and NMR structures of IL-8 yields backbone atomic r.m.s. differences of 0.99 and 1.28 A, respectively for individual monomers, and 1.08 and 1.82 A, respectively for the dimers (using MGSA residues 8 to 14 and 19 to 69). In general, the MGSA structure resembles the IL-8 X-ray structure more than it does the IL-8 NMR structure. At the tertiary (monomer) level the two main differences between the MGSA solution structure and IL-8 NMR structure involve the loops between residues 14 to 19 and between residues 30 to 38. At the quaternary (dimer) level the difference results from differing angles between the beta-strands which form the dimer interface, and is manifest as a different interhelical separation (distance of closest approach between the two helices is 15.3 A in the IL-8 NMR structure and 11.7 (+/- 0.4) A in the MGSA structure).

Two-dimensional NMR spectroscopy has been used to characterize the solution conformation of an atrial natriuretic peptide (ANP) variant which is selective for the human natriuretic peptide receptor A (NPR-A) relative to receptor C (NPR-C). The ANP mutant, containing six substitutions, has reduced flexibility in aqueous solution relative to wild-type ANP and allows the observation of sufficient NOE connectivities for structure determination by distance geometry and restrained molecular dynamics calculations. The solution conformation is reasonably well defined, having an average backbone atom rms deviation from the average coordinates of approximately 1.1 A for residues 7-27. The structure is consistent with available functional data and shows a spatial separation between known receptor binding determinants and residues found to be outside the hormone-receptor interface.

Recently, a 29-residue cyclic peptide was synthesized (TrPepz) that was reported to possess nearly the same catalytic activity and specificity as the pancreatic serine protease, trypsin, for hydrolysis of a small ester substrate, N-tosyl-L-arginine methyl ester (TAME), and small and large peptides [Atassi, M. Z. & Manshouri, T. (1993) Proc. Natl. Acad Sci. USA 90, 8282-8286]. To study these results we have resynthesized TrPepz and a related cyclic peptide reported to possess some trypsin-like activity. The authenticity of each peptide was confirmed by mass spectrometry, peptide sequencing, compositional analysis, and 1H NMR spectroscopy. However, neither peptide exhibited any detectable esterase activity or amidase activity under a variety of conditions tested. Molecular modeling studies indicated it was possible for TrPepz to be nearly superimposed upon the active site of trypsin. However, NMR experiments showed the structure of the cyclic peptide to be disordered. Thus, we were unable to confirm the results of Atassi and Manshouri. Our results are consistent with the view that serine protease activity depends not only on the presence of catalytic groups but also on their precise and stable alignment.

A novel protocol for isotopically labeling bacterially expressed proteins is presented. This method circumvents problems related to poor cell growth, commonly associated with the use of minimal labeled media, and problems with protein induction encountered, less commonly, when using enriched labeled media. The method involves initially growing the bacterial cells to high optical density in a commercially available enriched labeled medium. Following a suitable growth period, the cells are transferred to a different (minimal) labeled medium, appropriate for induction. The method is demonstrated using the protein melanoma growth stimulating activity (MGSA).

The solution structure of melanoma growth stimulating activity (MGSA) has been investigated using proton NMR spectroscopy. Sequential resonance assignments have been carried out, and elements of secondary structure have been identified on the basis of NOE, coupling constant, chemical shift, and amide proton exchange data. Long-range NOEs have established that MGSA is a dimer in solution. The secondary structure and dimer interface of MGSA appear to be similar to those found previously for the homologous chemokine interleukin-8 [Clore et al. (1990) Biochemistry 29, 1689-1696]. The MGSA monomer contains a three stranded anti-parallel beta-sheet arranged in a 'Greek-key' conformation, and a C-terminal alpha-helix (residues 58-69).

Almost complete assignments of the 1H, 15N, and aliphatic 13C resonances of the 62-residue N-terminal DNA-binding domain of GAL4 [GAL4(62)] have been obtained using a combination of two-dimensional homonuclear and two- and three-dimensional double- and triple-resonance heteronuclear NMR methods. The sequential NOE connectivities, amide proton exchange measurements, and 13C alpha chemical shift data indicate the presence of two short alpha-helices in the N-terminal half of the polypeptide. Residues 1-9 and 41-62 appear to be unstructured and flexible in solution. Analysis of the 13C alpha chemical shifts also revealed a significant downfield shift of approximately +3 ppm, relative to random-coil values, for the four nonbridging Zn(II) ligands, Cys 14, 21, 31, and 38. Interestingly, no such correlation was observed for the two bridging ligands, Cys 11 and 28. Preliminary structure calculations using a subset of distance restraints derived from three-dimensional 1H-15N and 1H-13C NOESY-HSQC spectra are consistent with the recently reported solution structures of Zn(II)2GAL4(7-49) [Kraulis, P., et al. (1992) Nature 356, 448-450] and of Cd(II)2GAL4(65) [Baleja, J. D., et al. (1992) Nature 356, 450-453].

Enzyme IIAglc and HPr are central regulatory and phosphocarrier proteins of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) of bacteria. During phosphoryl transfer from phosphoenolpyruvate to glucose, phosphate is transferred from HPr to enzyme IIAglc. In order to characterize the binding interfaces of the two proteins during phosphate transfer, 15N-edited and 15N-filtered NMR experiments have been recorded for the complex of enzyme IIAglc and HPr from Bacillus subtilis. Uniformly 15N-labeled enzyme IIAglc and nonlabeled HPr were used in these studies. Residues which undergo significant chemical shift changes upon complex formation have been identified for both proteins. The binding interfaces of the two proteins, suggested by the observed chemical shift changes, involve predominantly hydrophobic surfaces near the active site His-15 of HPr and the phosphoryl acceptor His-83 of IIAglc.

Enzyme IIA and HPr are central regulatory proteins of the bacterial phosphoenolpyruvate:sugar phosphotransferase (PTS) system. Three-dimensional structures of the glucose enzyme IIA domain (IIAglc) and HPr of Bacillus subtilis and Escherichia coli have been studied by both X-ray crystallography and Nuclear Magnetic Resonance (NMR) Spectroscopy. Phosphorylation of HPr of B. subtilis and IIAglc of E. coli have also been characterized by NMR spectroscopy. In addition, the binding interfaces of B. subtilis HPr and IIAglc have been identified from backbone chemical shift changes. This paper reviews these recent advances in the understanding of the three-dimensional structures of HPr and IIAglc and their interaction with each other.

The role of two "basic patch" residues, Arg-38 and His-62, in the catalytic function and anion-dependent activation of yeast 3-phosphoglycerate kinase (PGK) was investigated by site-directed mutagenesis. Steady-state kinetics and NMR experiments were conducted to characterize the functional properties and structural integrity of the R38A and H62A mutants. The results of these studies, in combination with earlier mutagenesis experiments, suggest that Arg-38 is the only catalytically essential residue among the conserved histidines and arginines of the basic patch. It appears that, similar to the remaining basic patch residues, His-62 is important for anion-dependent activation but not for enzyme activity. Cumulative evidence from this study and from previous mutagenesis experiments suggests that the basic patch region is in effect an extended anion binding site that encompasses both the catalytic and the general anion-binding site. It is proposed that substitution of any one of the basic patch residues results in an increased localization of the catalytic site. Substrate and product may still bind to this site, but a simultaneous binding of activatory anions, required for activation, has been impaired. NMR experiments suggest that the conformational changes observed upon binding of 3-PG to wild-type PGK are necessary for anion- and substrate-dependent activation.

The backbone dynamics of the uniformly 15N-labeled IIA domain of the glucose permease of Bacillus subtilis have been characterized using inverse-detected two-dimensional 1H-15N NMR spectroscopy. Longitudinal (T1) and transverse (T2) 15N relaxation time constants and steady-state (1H)-15N NOEs were measured, at a spectrometer proton frequency of 500 MHz, for 137 (91%) of the 151 protonated backbone nitrogens. These data were analyzed by using a model-free dynamics formalism to determine the generalized order parameter (S2), the effective correlation time for internal motions (tau e), and 15N exchange broadening contributions (Rex) for each residue, as well as the overall molecular rotational correlation time (tau m). The T1 and T2 values for most residues were in the ranges 0.45-0.55 and 0.11-0.15 s, respectively; however, a small number of residues exhibited significantly slower relaxation. Similarly, (1H)-15N NOE values for most residues were in the range 0.72-0.80, but a few residues had much smaller positive NOEs and some exhibited negative NOEs. The molecular rotational correlation time was 6.24 +/- 0.01 ns; most residues had order parameters in the range 0.75-0.90 and tau e values of less than ca. 25 ps. Residues found to be more mobile than the average were concentrated in three areas: the N-terminal residues (1-13), which were observed to be highly disordered; the loop from P25 to D41, the apex of which is situated adjacent to the active site and may have a role in binding to other proteins; and the region from A146 to S149. All mobile residues occurred in regions close to termini, in loops, or in irregular secondary structure.

Nearly complete assignment of the aliphatic 1H and 13C resonances of the IIAglc domain of Bacillus subtilis has been achieved using a combination of double- and triple-resonance three-dimensional (3D) NMR experiments. A constant-time 3D triple-resonance HCA(CO)N experiment, which correlates the 1H alpha and 13C alpha chemical shifts of one residue with the amide 15N chemical shift of the following residue, was used to obtain sequence-specific assignments of the 13C alpha resonances. The 1H alpha and amide 15N chemical shifts had been sequentially assigned previously using principally 3D 1H-15N NOESY-HMQC and TOCSY-HMQC experiments [Fairbrother, W. J., Cavanagh, J., Dyson, H. J., Palmer, A. G., III, Sutrina, S. L., Reizer, J., Saier, M. H., Jr., & Wright, P. E. (1991) Biochemistry 30, 6896-6907]. The side-chain spin systems were identified using 3D HCCH-COSY and HCCH-TOCSY spectra and were assigned sequentially on the basis of their 1H alpha and 13C alpha chemical shifts. The 3D HCCH and HCA(CO)N experiments rely on large heteronuclear one-bond J couplings for coherence transfers and therefore offer a considerable advantage over conventional 1H-1H correlation experiments that rely on 1H-1H 3J couplings, which, for proteins the size of IIAglc (17.4 kDa), may be significantly smaller than the 1H line widths. The assignments reported herein are essential for the determination of the high-resolution solution structure of the IIAglc domain of B. subtilis using 3D and 4D heteronuclear edited NOESY experiments; these assignments have been used to analyze 3D 1H-15N NOESY-HMQC and 1H-13C NOESY-HSQC spectra and calculate a low-resolution structure [Fairbrother, W. J., Gippert, G. P., Reizer, J., Saier, M. H., Jr., & Wright, P. E. (1992) FEBS Lett. 296, 148-152].

A low resolution solution structure of the IIA domain of the Bacillus subtilis phosphoenolpyruvate-sugar phosphotransferase system (PTS) glucose permease has been determined using 945 inter-residue and 724 intra-residue distance constraints derived from three-dimensional 15N and 13C edited NOESY spectra. A total of 15 structures was generated using distance geometry calculations. The protein is comprised of 13 beta-strands forming an antiparallel beta-barrel. The average backbone atomic RMS deviation about the average distance geometry structure for the beta-sheet residues is 1.1 A. The conformations of the loop regions between the beta-strands are less well determined. Backbone distance constraints obtained during the process of sequential assignment were insufficient to correctly calculate the polypeptide fold.

Two new protocols for the three-dimensional, triple resonance, constant-time HCA(CO)N NMR experiment are presented that significantly increase the experimental resolution attainable in the C alpha frequency dimension. Experimental verification of the new experiments is provided by spectra of the IIA domain of glucose permease from Bacillus subtilis.

The enzyme IIIglc-like domain of Bacillus subtilis IIglc (IIIglc, 162 residues, 17.4 kDa) has been cloned and overexpressed in Escherichia coli. Sequence-specific assignment of the backbone 1H and 15N resonances has been carried out with a combination of homonuclear and heteronuclear two-dimensional and heteronuclear three-dimensional (3D) NMR spectroscopy. Amide proton solvent exchange rate constants have been determined from a series of 1H-15N heteronuclear single-quantum coherence (HSQC) spectra acquired following dissolution of the protein in D2O. Major structural features of IIIglc have been inferred from the pattern of short-, medium- and long-range NOEs in 3D heteronuclear 1H nuclear Overhauser effect 1H-15N multiple-quantum coherence (3D NOESY-HMQC) spectra, together with the exchange rate constants. IIIglc contains three antiparallel beta-sheets comprised of eight, three, and two beta-strands. In addition, five beta-bulges were identified. No evidence of regular helical structure was found. The N-terminal 15 residues of the protein appear disordered, which is consistent with their being part of the Q-linker that connects the C-terminal enzyme IIIglc-like domain to the membrane-bound IIglc domain. Significantly, two histidine residues, His 68 and His 83, which are important for phosphotransferase function, are found from NOE measurements to be in close proximity at the ends of adjacent strands in the major beta-sheet.

1H-NMR measurements were made of solutions of yeast phosphoglycerate kinase containing the nucleotide substrate, ATP, and Mg2+ in varying concentrations in order to investigate the affect that the metal ion has on the mode of ATP binding to the enzyme. From the change in the chemical shifts of the 'basic-patch' histidine resonances (His62, His167 and His170) and the nucleotide C8H, C2H and C1'H resonances it is apparent that there are at least two ATP-binding sites on the enzyme. Downfield shifts observed for the above histidine resonances at low nucleotide/enzyme molar ratios indicates that the primary binding site involves electrostatic interactions between the nucleotide triphosphate chain and the basic-patch region of the N-terminal domain. The secondary binding site is shown to involve predominantly hydrophobic interactions between the adenosine moiety and the protein. Evidence from previous two-dimensional NMR experiments [Fairbrother et al. (1990) Eur. J. Biochem. 190, 161-169] suggests that the secondary site is equivalent to the crystallographically observed catalytic site. The affinity of the catalytic site is increased relative to the primary electrostatic site with increasing Mg2+ concentration. The possible importance of these observations in the regulation of this enzyme in vivo are discussed.

Anion binding to yeast phosphoglycerate kinase has been investigated using 1H-NMR spectroscopy. The use of anionic paramagnetic probes. [Cr(CN)6]3- and [Fe(CN)6]3-, has enabled the location of the primary anion binding site in the 'basic-patch' region of the amino-terminal domain. The anions interact most closely with Arg-65 and Arg-168. The binding of these and a variety of other anions to this site is directly competitive with the binding of the substrate, 3-phosphoglycerate. Binding of 3-phosphoglycerate and 1.3-bisphosphoglycerate is, however, stronger than expected on the basis of anionic charge and causes conformational changes in the protein not seen with any of the other simple spherical anions investigated. This must be part, at least, of the substrate specificity. Evidence for a secondary anion binding site involving the side chains of surface lysine residues is also presented. It is suggested that the primary anion site is responsible for the observed activation by anions at low concentrations while the secondary site leads to inhibition at higher anion concentrations. The kinetics fit these deductions and a scheme for kinase activity is presented.

Site-directed mutagenesis has been used to produce a mutant form of yeast phosphoglycerate kinase (PGK) in which the 'basic patch' residue His 62 has been replaced by a glutamine residue. Using 1H-NMR spectroscopy, it was found that 3-phosphoglycerate (3-PG) binding to the mutant protein induces the same conformational effects as for wild-type PGK, although the affinity was reduced by 2- to 3-fold. Kinetic studies show both Km for 3-PG and Vmax to be increased by approximately 2-fold relative to the wild-type enzyme. These data are consistent with the suggestion that His 62 assists in the binding of the substrate to the enzyme.

One- and two-dimensional proton NMR studies have been carried out on yeast phosphoglycerate kinase (Mr approximately 45,000) in order to identify amino-acid spin systems and obtain sequence-specific assignments. A number of sequence-specific assignments have been made using a combination of structural information contained in nuclear Overhauser effect spectra and X-ray crystallographic data. The results of substrate binding studies (both 3-phosphoglycerate and Mg.ATP), which indicate mutual reorientation of certain assigned aromatic residues in the inter-domain region of the protein, are discussed.

The structural integrity and substrate binding properties of the two genetically engineered domains of yeast phosphoglycerate kinase were investigated using one- and two-dimensional nuclear magnetic resonance techniques. Both domains were found to fold with regions of native-like structure, with the N-domain showing greater conformational flexibility than the C-domain. The 'basic patch' region of the N-domain is, however, clearly perturbed by removal of the C-domain. This is most likely due to the absence of stabilizing interactions between the C-terminal peptide (including alpha-helices XIII and XIV) and the N-domain. The C-domain is able to bind nucleotide with an affinity only three times less than that of the native protein.

Site-specific mutants of yeast phosphoglycerate kinase have been produced in order to investigate the roles of the 'basic-patch' residues, arginine 168 and histidine 170. The fully-conserved residue, arginine 168, has been replaced with a lysine (R168K) and a methionine (R168M) residue, while the non-conserved histidine 170 has been replaced with an aspartate (H170D). Comparison of the 500-MHz 1H-NMR spectra of the mutant proteins with that of wild-type phosphoglycerate kinase shows that the overall fold of the mutants remains essentially unaltered from that of the native enzyme. Results of NOE experiments indicate that there are only very minor changes in structure in the vicinity of the mutations. These mutations have also led to firm sequence-specific resonance assignments to histidines 62, 167 and 170. NMR studies of 3-phosphoglycerate binding show that decreasing the positive charge in the sequence 168-170 reduces the binding of this substrate (by about 15-fold and 4-fold for mutants R168M and H170D respectively). Mutant R168K binds 3-phosphoglycerate with an affinity about twofold less than that of the native enzyme. Significantly, the activity of mutant H170D, measured at saturating substrate concentrations, is unchanged from that of the wild-type enzyme. This indicates that this residue is not of major importance in the binding or reaction of 3-phosphoglycerate. The observation is in agreement with results obtained for the wild-type enzyme, which indicate that 3-phosphoglycerate interacts most strongly with histidine 62 and least strongly with histidine 170, as would be predicted from the X-ray crystal structure. Substitution of positively charged arginine 168 with neutral methionine (or positively charged lysine) does not cause a detectable change in the pKa values of the neighbouring histidine groups, in as much as they remain below 3. The results reported here indicate that the observed reduction in catalytic efficiency relates less to direct electrostatic effects than to the mutants' inability to undergo 3-phosphoglycerate-induced conformational changes.

The binding of a series of inhibitors to the enzyme phosphoglycerate kinase has been studied using NMR to uncover the binding sites and the effects of binding on the protein conformation. The very effective inhibitor, Suramin, causes the most pronounced changes. The design of inhibitors for mobile proteins is discussed.