A sequence motif that is Similar to Ubiquitin (SUb) has been identified in the Saccharomyces cerevisiae ubiquitin-specific protease Ubp6. SUb is conserved in all known Ubp6 homologues from a spectrum of eukaryotic species and is also present in a group of hypothetical proteins of unknown function (Unk1-3) present in sequence databases. An N-terminal deletion mutant of Ubp6 that lacks SUb is still capable of cleaving α-linked ubiquitin fusions, suggesting that SUb forms a separate domain to the catalytic core of Ubp6 and demonstrating that it is not required for in vitro cleavage activity. A homology model of the 78 N-terminal amino acids of human Ubp6, based on the known fold of ubiquitin, is presented. In human Ubp6, SUb shares only 20% sequence identity with ubiquitin. Even weaker similarity occurs between S. cerevisiae SUb and ubiquitin. The homology model supports a ubiquitin-like fold for SUb and suggests that two conserved Lys residues, corresponding to Lys48 and Lys63 of ubiquitin, are functionally important.

Urea-induced unfolding of Escherichia coli citrate synthase occurs in two phases, as monitored by circular dichroism at 222 nm (measuring secondary structure) or by tryptophan fluorescence. In this paper we characterize the intermediate state, which retains about 40% of the ellipticity of the native state, and is stable between 2.5 M and 5.5 M urea, approximately. This intermediate binds significant amounts of the probe for hydrophobic surfaces, anilinonaphthalene sulfonate, but forms aggregates at least as high as an octamer, as shown by transverse urea gradient polyacrylamide electrophoresis. Thermal denaturation of E. coli citrate synthase also produces an intermediate at temperatures near 60 °C, which also retains about 40% of the native ellipticity and forms aggregates, as measured by electrospray-ionization/time-of-flight mass spectrometry. We have used a collection of “cavity-forming” mutant proteins, in which bulky buried hydrophobic residues are replaced by alanines, to explore the nature of the intermediate state further. A certain amount of these mutant proteins shows a destabilized intermediate, as measured by the urea concentration range in which the intermediate is observed. These mutants are found in parts of the citrate synthase sequence that, in a native state, form helices G, M, N, Q, R, and S. From this and other evidence, it is argued that the intermediate state is an aggregated state in which these six helices, or parts of them, remain folded, and that formation of this intermediate is also likely to be a key step in the folding of E. coli citrate synthase.

Computer simulations of protein models on lattices have been widely used as an aid in the study of protein folding process. Following the suggestion of Raghunathan and Jernigan (1997, Protein Sci 6:2072–2083) that the cubo-octahedral lattice can allow a more realistic representation of proteins than other lattices, we propose here the use of a new set of internal coordinates θ for the description of a protein model on this lattice. An easy procedure for the conversion of the θ coordinates to the Cartesian coordinates is also described. When the Contact Interaction approach, already proposed by us for simulations on square or cubic lattices, was applied to the cubo-octahedral lattice, the system obeyed the correct thermodynamics derived from the definition of energy. Thus, lattice simulations of protein models, in which secondary structure elements such as α-helices or β-strands can be easily identifiable, can be performed.

An NIH Grant number was inadvertently omitted from the acknowledgments printed on p. 2063 of the above referenced article (Volume 8, Number 10, 1999, pp. 2055–2064). The complete text of the Acknowledgments is reprinted below (the online edition of the article has been amended to correct for the omissions):

Acknowledgments

The author is indebted to Dr. David Atkinson for his support and help at all stages of this work and for a thorough reading of the manuscript; to Dr. Mary T. Walsh for her encouragement, help with the CD maintenance, and helpful comments on the manuscript; to Dr. G. Graham Shipley for critical reading on the manuscript; and to Michael Gigliotti for technical support. This work was supported by Grant-in-Aid 13-512-956 from the American Heart Association (Massachusetts Affiliate) and NIH Grant HL61429 to O.G., and by NIH Grant HL26335 (D.M. Smith, P.I.).

NMR offers the possibility of accurate secondary structure for proteins that would be too large for structure determination. In the absence of an X-ray crystal structure, this information should be useful as an adjunct to protein fold recognition methods based on low resolution force fields. The value of this information has been tested by adding varying amounts of artificial secondary structure data and threading a sequence through a library of candidate folds. Using a literature test set, the threading method alone has only a one-third chance of producing a correct answer among the top ten guesses. With realistic secondary structure information, one can expect a 60–80% chance of finding a homologous structure. The method has then been applied to examples with published estimates of secondary structure. This implementation is completely independent of sequence homology, and sequences are optimally aligned to candidate structures with gaps and insertions allowed. Unlike work using predicted secondary structure, we test the effect of differing amounts of relatively reliable data.

Starch phosphorylase from Corynebacterium callunae is a dimeric protein in which each mol of 90 kDa subunit contains 1 mol pyridoxal 5′-phosphate as an active-site cofactor. To determine the mechanism by which phosphate or sulfate ions bring about a greater than 500-fold stabilization against irreversible inactivation at elevated temperatures (≥50 °C), enzyme/oxyanion interactions and their role during thermal denaturation of phosphorylase have been studied. By binding to a protein site distinguishable from the catalytic site with dissociation constants of Ksulfate = 4.5 mM and Kphosphate ≈ 16 mM, dianionic oxyanions induce formation of a more compact structure of phosphorylase, manifested by (a) an increase by about 5% in the relative composition of the α-helical secondary structure, (b) reduced 1H/2H exchange, and (c) protection of a cofactor fluorescence against quenching by iodide. Irreversible loss of enzyme activity is triggered by the release into solution of pyridoxal 5′-phosphate, and results from subsequent intermolecular aggregation driven by hydrophobic interactions between phosphorylase subunits that display a temperature-dependent degree of melting of secondary structure. By specifically increasing the stability of the dimer structure of phosphorylase (probably due to tightened intersubunit contacts), phosphate, and sulfate, this indirectly (1) preserves a functional active site up to ≈50 °C, and (2) stabilizes the covalent protein cofactor linkage up to ≈70 °C. The effect on thermostability shows a sigmoidal and saturatable dependence on the concentration of phosphate, with an apparent binding constant at 50 °C of ≈25 mM. The extra stability conferred by oxyanion–ligand binding to starch phosphorylase is expressed as a dramatic shift of the entire denaturation pathway to a ≈20 °C higher value on the temperature scale.

The binding of l-serine to phosphoglycerate dehydrogenase from Escherichia coli displays elements of both positive and negative cooperativity. At pH 7.5, ∼2 mol of serine are bound per mole of tetrameric enzyme. A substantial degree of positive cooperativity is seen for the binding of the second ligand, but the binding of the third and fourth ligand display substantial negative cooperativity. The data indicate a state of ∼50% inhibition when only one serine is bound and ∼80–90% inhibition when two serines are bound. This is consistent with the tethered domain hypothesis that has been presented previously. Comparison of the data derived directly from binding stoichiometry to the binding constants determined from the best fit to the Adair equation, produce a close agreement, and reinforce the general validity of the derived binding constants. The data also support the conclusion that the positive cooperativity between the binding to the first and second site involves binding sites at opposite interfaces over 110 Å apart. Thus, an order of binding can be envisioned where the binding of the first ligand initiates a conformational transition that allows the second ligand to bind with much higher affinity at the opposite interface. This is followed by the third ligand, which binds with lesser affinity to one of the two already occupied interfaces, and in so doing, completes a global conformational transition that produces maximum inhibition of activity and an even lower affinity for the fourth ligand, excluding it completely. Thus, maximal inhibition is accomplished with less than maximal occupancy of effector sites through a mechanism that displays strong elements of both positive and negative cooperativity.

To test, at the level of individual amino acids, the conformation of an exchangeable apolipoprotein in aqueous solution and in the presence of an osmolyte trimethylamine-N-oxide (TMAO), six synthetic peptide analogues of human apolipoprotein C-1 (apoC-1, 57 residues) containing point mutations in the predicted α-helical regions were analyzed by circular dichroism (CD). The CD spectra and the melting curves of the monomeric wild-type and plasma apoC-1 in neutral low-salt solutions superimpose, indicating 31 ± 4% α-helical structure at 22 °C that melts reversibly with Tm,WT = 50 ± 2 °C and van't Hoff enthalpy ΔHv,WT(Tm) = 18 ± 2 kcal/mol. G15A substitution leads to an increased α-helical content of 42 ± 4% and an increased Tm,G15A = 57 ± 2 °C, which corresponds to stabilization by δΔGapp = +0.4 ± 1.5 kcal/mol. G15P mutant has ∼20% α-helical content at 22 °C and unfolds with low cooperativity upon heating to 90 °C. R23P and T45P mutants are fully unfolded at 0–90 °C. In contrast, Q31P mutation leads to no destabilization or unfolding. Consequently, the R23 and T45 locations are essential for the stability of the cooperative α-helical unit in apoC-1 monomer, G15 is peripheral to it, and Q31 is located in a nonhelical linker region. Our results suggest that Pro mutagenesis coupled with CD provides a tool for assigning the secondary structure to protein groups, which should be useful for other self-associating proteins that are not amenable to NMR structural analysis in aqueous solution. TMAO induces a reversible cooperative coil-to-helix transition in apoC-1, with the maximal α-helical content reaching 74%. Comparison with the maximal α-helical content of 73% observed in lipid-bound apoC-1 suggests that the TMAO-stabilized secondary structure resembles the functional lipid-bound apolipoprotein conformation.

The solution structure and backbone dynamics of Cu(I) pseudoazurin, a 123 amino acid electron transfer protein from Paracoccus pantotrophus, have been determined using NMR methods. The structure was calculated to high precision, with a backbone RMS deviation for secondary structure elements of 0.35 ± 0.06 Å, using 1,498 distance and 55 torsion angle constraints. The protein has a double-wound Greek-key fold with two α-helices toward its C-terminus, similar to that of its oxidized counterpart determined by X-ray crystallography. Comparison of the Cu(I) solution structure with the X-ray structure of the Cu(II) protein shows only small differences in the positions of some of the secondary structure elements. Order parameters S2, measured for amide nitrogens, indicate that the backbone of the protein is rigid on the picosecond to nanosecond timescale.

Two high resolution crystal structures of Escherichia coli alkaline phosphatase (AP) in the presence of phosphonate inhibitors are reported. The phosphonate compounds, phosphonoacetic acid (PAA) and mercaptomethylphosphonic acid (MMP), bind competitively to AP with dissociation constants of 5.5 and 0.6 mM, respectively. The structures of the complexes of AP with PAA and MMP were refined at high resolution to crystallographic R-values of 19.0 and 17.5%, respectively. Refinement of the AP-inhibitor complexes was carried out using X-PLOR. The final round of refinement was done using SHELXL-97. Crystallographic analyses of the inhibitor complexes reveal different binding modes for the two phosphonate compounds. The significant difference in binding constants can be attributed to these alternative binding modes observed in the high resolution X-ray structures. The phosphinyl group of PAA coordinates to the active site zinc ions in a manner similar to the competitive inhibitor and product inorganic phosphate. In contrast, MMP binds with its phosphonate moiety directed toward solvent. Both enzyme-inhibitor complexes exhibit close contacts, one of which has the chemical and geometrical potential to be considered an unconventional hydrogen bond of the type C-H···X.

The binding capacity of the L-leucine receptor from Escherichia coli was measured with L-phenylalanine and 4-fluoro-L-phenylalanine as substrates by fluorescence. The apparent dissociation constants (KD) for L-leucine, L-phenylalanine, and 4-fluoro-L-phenylalanine are 0.40, 0.18, and 0.26 respectively. 19F NMR data show protein-induced shifts for the 4-fluoro-L-phenylalanine peak and 3-fluoro-L-phenylalanine when receptor is present. Evidence points to the binding of only the L-isomers of these fluorine analogs.

Physicochemical properties are potentially useful in predicting functional differences between aligned protein subfamilies. We present a method that considers physicochemical properties from ancestral sequences predicted to have given rise to the subfamilies of interest by gene duplication. Comparison between two map kinases subfamilies, p38 and ERK, revealed a region that had an excess of change in properties after gene duplication followed by conservation within the two subfamilies. This region corresponded to that experimentally defined as important for substrate and pathway specificity. The derived scores for the region of interest were found to differ significantly in their distribution compared to the rest of the protein when the Kolmogorov–Smirnov test was applied (p = 0.005). Thus, the incorporation of ancestral physicochemical properties is useful in predicting functional differences between protein subfamilies. In addition, the method was applied to the MKK and MAPK components of the p38 and JNK pathways. These proteins showed a similar pattern in their evolution and regions predicted to confer functional differences are discussed.

The role of the switch II region in the conformational transition of activation of Ha-ras-p21 has been investigated by mutating residues predicted to act as hinges for the conformational transition of this loop (Ala59, Gly60, and Gly75) (Diaz JF, Wroblowski B, Schlitter J, Engelborghs Y, 1997, Proteins 28:434–451), as well as mutating the catalytic residue Gln61. The proposed mutations of the hinge residues decrease the rate of the conformational transition of activation as measured by the binding of BeF3− to the GDP-p21 complex. Also, the thermodynamic parameters of the binding reaction are altered by a factor between three and five, depending on the temperature. (Due to changes in activation and reaction enthalpies, partially compensated by entropy changes.) The control mutation Q61H in which only the catalytic residue is changed has only a limited effect on the kinetic rate constants of the conformational transition and on the thermodynamic parameters of the reaction.

The fact that mutations of the hinge residues of the switch II region affect both the binding of the phosphate analog and the conformational transition of activation indicates that the switch II is implicated both in the early and the late states of the transition.

Phosphoglucose isomerase (EC 5.3.1.9) catalyzes the interconversion of d-glucopyranose-6-phosphate and d-fructofuranose-6-phosphate by promoting an intrahydrogen transfer between C1 and C2. A conserved histidine exists throughout all phosphoglucose isomerases and was hypothesized to be the base catalyzing the isomerization reaction. In the present study, this conserved histidine, His311, of the enzyme from Bacillus stearothermophilus was subjected to mutational analysis, and the mutational effect on the inactivation kinetics by N-bromoacetylethanolamine phosphate was investigated. The substitution of His311 with alanine, asparagine, or glutamine resulted in the decrease of activity, in kcat/KM, by a factor of 103, indicating the importance of this residue. N-bromoacetylethanolamine phosphate inactivated irreversibly the activity of wild-type phosphoglucose isomerase; however, His311 → Ala became resistant to this inhibitor, indicating that His311 is located in the active site and is responsible for the inactivation of the enzyme by this active site-directed inhibitor. The pKa of His311 was estimated to be 6.31 according to the pH dependence of the inactivation. The proximity of this value with the pKa value of 6.35, determined from the pH dependence of kcat/KM, supports a role of His311 as a general base in the catalysis.

An α-helix and a β-strand are said to be interactively packed if at least one residue in each of the secondary structural elements loses 10% of its solvent accessible contact area on association with the other secondary structural element. An analysis of all such 5,975 nonidentical α/β units in protein structures, defined at ≤2.5 Å resolution, shows that the interaxial distance between the α-helix and the β-strand is linearly correlated with the residue-dependent function, log[(V/nda)/n-int], where V is the volume of amino acid residues in the packing interface, nda is the normalized difference in solvent accessible contact area of the residues in packed and unpacked secondary structural elements, and n-int is the number of residues in the packing interface. The β-sheet unit (βu), defined as a pair of adjacent parallel or antiparallel hydrogen-bonded β-strands, packing with an α-helix shows a better correlation between the interaxial distance and log(V/nda) for the residues in the packing interface. This packing relationship is shown to be useful in the prediction of interaxial distances in α/β units using the interacting residue information of equivalent α/β units of homologous proteins. It is, therefore, of value in comparative modeling of protein structures.

The human small GTPase, RhoA, expressed in Saccharomyces cerevisiae is post-translationally processed and, when co-expressed with its cytosolic inhibitory protein, RhoGDI, spontaneously forms a heterodimer in vivo. The RhoA/RhoGDI complex, purified to greater than 98% at high yield from the yeast cytosolic fraction, could be stoichiometrically ADP-ribosylated by Clostridium botulinum C3 exoenzyme, contained stoichiometric GDP, and could be nucleotide exchanged fully with [3H]GDP or partially with GTP in the presence of submicromolar Mg2+. The GTP-RhoA/RhoGDI complex hydrolyzed GTP with a rate constant of 4.5 × 10−5 s−1, considerably slower than free RhoA. Hydrolysis followed pseudo-first-order kinetics indicating that the RhoA hydrolyzing GTP was RhoGDI associated. The constitutively active G14V-RhoA mutant expressed as a complex with RhoGDI and purified without added nucleotide also bound stoichiometric guanine nucleotide: 95% contained GDP and 5% GTP. Microinjection of the GTP-bound G14V-RhoA/RhoGDI complex (but not the GDP form) into serum-starved Swiss 3T3 cells elicited formation of stress fibers and focal adhesions. In vitro, GTP-bound-RhoA spontaneously translocated from its complex with RhoGDI to liposomes, whereas GDP-RhoA did not. These results show that GTP-triggered translocation of RhoA from RhoGDI to a membrane, where it carries out its signaling function, is an intrinsic property of the RhoA/RhoGDI complex that does not require other protein factors or membrane receptors.

B*2701 differs from all other HLA-B27 subtypes of known peptide specificity in that, among its natural peptide ligands, arginine is not the only allowed residue at peptide position 2. Indeed, B*2701 is unique in binding many peptides with Gln2 in vivo. However, the mutation (Asp74Tyr) responsible for altered selectivity is far away from the B pocket of the peptide binding site to which Gln/Arg2 binds. Here, we present a model that explains this effect. It is proposed that a new rotameric state of the conserved Lys70 is responsible for the unique B*2701 binding motif. This side chain should be either kept away from pocket B through its interaction with Asp74 in most HLA-B27 subtypes, or switched to this pocket if residue 74 is Tyr as in B*2701. Involvement of Lys70 in pocket B would thus allow binding of peptides with Gln2. Binding of Arg2-containing peptides to B*2701 is also possible because Lys70 could adopt another conformation, H-bonded to Asn97, which preserves the same binding mode of Arg2 as in B*2705. This model was experimentally validated by mutating Lys70 into Ala in B*2701. Edman sequencing of the B*2701(K70A) peptide pool showed only Arg2, characteristic of HLA-B27-bound peptides, and no evidence for Gln2. This supports the computational model and demonstrates that allowance of B*2701 for peptides with Gln2 is due to the long-range effect of the polymorphic residue 74 of HLA-B27, by inducing a conformational switch of the conserved Lys70.

The tumor suppressor function of the wild-type p53 protein is transdominantly inhibited by tumor-derived mutant p53 proteins. Such transdominant inhibition limits the prospects for gene therapy approaches that aim to introduce wild-type p53 into cancer cells. The molecular mechanism for transdominant inhibition involves sequestration of wild-type p53 subunits into inactive wild-type/mutant hetero-tetramers. Thus, p53 proteins, whose oligomerization specificity is altered so they cannot interact with tumor-derived mutant p53, would escape transdominant inhibition. Aided by the known three-dimensional structure of the p53 tetramerization domain and by trial and error we designed a novel domain with seven amino acid substitutions in the hydrophobic core. A full-length p53 protein bearing this novel domain formed homo-tetramers and had tumor suppressor function, but did not hetero-oligomerize with tumor-derived mutant p53 and resisted transdominant inhibition. Thus, hydrophobic core residues influence the oligomerization specificity of the p53 tetramerization domain.

Surveys of protein crystal structures have revealed that amino acids show unique structural preferences for the N1, N2, and N3 positions in the first turn of the α-helix. We have therefore extended helix-coil theory to include statistical weights for these locations. The helix content of a peptide in this model is a function of N-cap, C-cap, N1, N2, N3, C1, and helix interior (N4 to C2) preferences. The partition function for the system is calculated using a matrix incorporating the weights of the fourth residue in a hexamer of amino acids and is implemented using a FORTRAN program. We have applied the model to calculate the N1 preferences of Gln, Val, Ile, Ala, Met, Pro, Leu, Thr, Gly, Ser, and Asn, using our previous data on helix contents of peptides Ac-XAKAAAAKAAGY-CONH2. We find that Ala has the highest preference for the N1 position. Asn is the most unfavorable, destabilizing a helix at N1 by at least 1.4 kcal mol−1 compared to Ala. The remaining amino acids all have similar preferences, 0.5 kcal mol−1 less than Ala. Gln, Asn, and Ser, therefore, do not stabilize the helix when at N1.

This work experimentally confirms the pathway of activation of Ha-ras-p21, which was calculated by the method of Targeted Molecular Dynamics (TMD) (Díaz JF, Wroblowski B, Schlitter J, Engelborghs Y, 1997a, Proteins Struct Funct Genet 28:434–451). The process can be studied experimentally by analyzing the binding of BeF3− to the GDP complex of the active fluorescent mutant Y32W (Díaz JF, Sillen A, Engelborghs Y, 1997b, J Biol Chem 227:23138–23143). Two mutants, V29G and I36G, have been constructed at both sides of the effector loop of the active fluorescent mutant. This was done to check the proposed reaction pathway and to provide further insight into the mechanism of the activation of ras proteins. Both mutations accelerate the conformational isomerization with two orders of magnitude, demonstrating convincingly the role of these residues as hinges of the effector loop in one or more of the transitions of the conformational change. These results provide experimental support to the pathway calculated by TMD analysis.