α2-Macroglobulin (α2M) is a major carrier of transforming growth factor-β (TGF-β) in vitro and in vivo. By screening glutathione S-transferase (GST) fusion proteins with overlapping sequences, we localized the TGF-β-binding site to aa 700–738 of the mature human α2M subunit. In separate experiments, we screened overlapping synthetic peptides corresponding to aa 696–777 of α2M and identified a single 16-mer (718–733) that binds TGF-β1. Platelet-derived growth factor-BB (PDGF-BB) bound to the same peptide, even though TGF-β and PDGF-BB share almost no sequence identity. The sequence of the growth factor-binding peptide, WDLVVVNSAGVAEVGV, included a high proportion of hydrophobic amino acids. The analogous peptide from murinoglobulin, a human α2M homologue that does not bind growth factors, contained only three nonconservative amino acid substitutions; however, the MUG peptide failed to bind TGF-β1 and PDGF-BB. These results demonstrate that a distinct and highly-restricted site in α2M, positioned near the C-terminal flank of the bait region, mediates growth factor binding. At least part of the growth factor-binding site is encoded by exon 18 of the α2M gene, which is notable for a 5′ splice site polymorphism that has been implicated in Alzheimer's Disease.

The amino acid sequence of the small copper protein auracyanin A isolated from the thermophilic photosynthetic green bacterium Chloroflexus aurantiacus has been determined to be a polypeptide of 139 residues. His58, Cys123, His128, and Met132 are spaced in a way to be expected if they are the evolutionary conserved metal ligands as in the known small copper proteins plastocyanin and azurin. Secondary structure prediction also indicates that auracyanin has a general β-barrel structure similar to that of azurin from Pseudomonas aeruginosa and plastocyanin from poplar leaves. However, auracyanin appears to have sequence characteristics of both small copper protein sequence classes. The overall similarity with a consensus sequence of azurin is roughly the same as that with a consensus sequence of plastocyanin, namely 30.5%. We suggest that auracyanin A, together with the B forms, is the first example of a new class of small copper proteins that may be descendants of an ancestral sequence to both the azurin proteins occurring in prokaryotic nonphotosynthetic bacteria and the plastocyanin proteins occurring in both prokaryotic cyanobacteria and eukaryotic algae and plants. The N-terminal sequence region 1–18 of auracyanin is remarkably rich in glycine and hydroxy amino acids, and required mass spectrometric analysis to be determined. The nature of the blocking group X is not yet known, although its mass has been determined to be 220 Da. The auracyanins are the first small blue copper proteins found and studied in anoxygenic photosynthetic bacteria and are likely to mediate electron transfer between the cytochrome bc1 complex and the photosynthetic reaction center.

The moderate thermophilic eubacterium Alicyclobacillus (formerly Bacillus) acidocaldarius expresses a thermostable carboxylesterase (esterase 2) belonging to the hormone-sensitive lipase (HSL)-like group of the esterase/lipase family. Based on secondary structures predictions and a secondary structure-driven multiple sequence alignment with remote homologous protein of known three-dimensional (3D) structure, we previously hypothesized for this enzyme the α/β-hydrolase fold typical of several lipases and esterases and identified Ser155, Asp252, and His282 as the putative members of the catalytic triad. In this paper we report the construction of a 3D model for this enzyme based on the structure of mouse acetylcholinesterase complexed with fasciculin. The model reveals the topological organization of the fold corroborating our predictions. As regarding the active-site residues, Ser155, Asp252, and His282 are located close to each other at hydrogen bond distances. Their catalytic role was here probed by biochemical and mutagenic studies. Moreover, on the basis of the secondary structure-driven multiple sequence alignment and the 3D structural model, a residue supposed important for catalysis, Gly84, was mutated to Ser. The activity of the mutated enzyme was drastically reduced. We propose that Gly84 is part of a putative “oxyanion hole” involved in the stabilization of the transition state similar to the C group of the esterase/lipase family.

GRASS (Graphical Representation and Analysis of Structures Server), a new web-based server, is described. GRASS exploits many of the features of the GRASP program and is designed to provide interactive molecular graphics and quantitative analysis tools with a simple interface over the World-Wide Web. Using GRASS, it is now possible to view many surface features of biological macromolecules on either standard workstations used in macromolecular analysis or personal computers. The result is a World-Wide Web-based, platform-independent, easily used tool for macromolecular visualization and structure analysis.

An expanded, highly dynamic denatured state of staphylococcal nuclease exhibits a native-like topology in the apparent absence of tight packing and fixed hydrogen bonds (Gillespie JR, Shortle D, 1997, J Mol Biol 268:158–169, 170–184). To address the physical basis of the long-range spatial ordering of this molecule, we probe the effects of perturbations of the sequence and solution conditions on the local chain dynamics of a denatured 101-residue fragment that is missing the first three beta strands. Structural interactions between chain segments are inferred from correlated changes in the motional behavior of residues monitored by 15N NMR relaxation measurements. Restoration of the sequence corresponding to the first three beta strands significantly increases the average order of all chain segments that form the five strand beta barrel including loops but has no effect on the carboxy terminal 30 residues. Addition of the denaturing salt sodium perchlorate enhances ordering over the entire sequence of this fragment. Analysis of seven different substitution mutants points to a complex set of interactions between the hydrophobic segment corresponding to beta strand 5 and the remainder of the chain. General patterns in the data suggest there is a hierarchy of native-like interactions that occur transiently in the denatured state and are consistent with the overall topology of the denatured state ensemble being determined by many coupled local interactions rather than a few highly specific long-range interactions.

We have reported variants of the M13 bacteriophage major coat protein (P8) that enable high copy display of monomeric and oligomeric proteins, such as human growth hormone and steptavidin, on the surface of phage particles (Sidhu SS, Weiss GA, Wells JA. 2000. High copy display of large proteins on phage for functional selections. J Mol Biol 296:487–495). Here, we explore how an optimized P8 variant (opti-P8) could evolve the ability to efficiently display a protein fused to its N-terminus. Reversion of individual opti-P8 residues back to the wild-type P8 residue identifies a limited set of hydrophobic residues responsible for the high copy protein display. These hydrophobic amino acids bracket a conserved hydrophobic face on the P8 alpha helix thought to be in contact with the phage coat. Mutations additively combine to promote high copy protein display, which was further enhanced by optimization of the linker between the phage coat and the fusion protein. These data are consistent with a model in which protein display-enhancing mutations allow for better packing of the fusion protein into the phage coat. The high tolerance for phage coat protein mutations observed here suggests that filamentous phage coat proteins could readily evolve new capabilities.

The native form of inhibitory serpins (serine protease inhibitors) is not in the thermodynamically most stable state but in a metastable state, which is critical to inhibitory functions. To understand structural basis and functional roles of the native metastability of inhibitory serpins, we have been characterizing stabilizing mutations of human α1-antitrypsin, a prototype inhibitory serpin. One of the sites that has been shown to be critical in stability and inhibitory activity of α1-antitrypsin is Lys335. In the present study, detailed roles of this lysine were analyzed by assessing the effects of 13 different amino acid substitutions. Results suggest that size and architect of the side chains at the 335 site determine the metastability of α1-antitrypsin. Moreover, factors such as polarity and flexibility of the side chain at this site, in addition to the metastability, seem to be critical for the inhibitory activity. Substitutions of the lysine at equivalent positions in two other inhibitory serpins, human α1-antichymotrypsin and human antithrombin III, also increased stability and decreased inhibitory activity toward α-chymotrypsin and thrombin, respectively. These results and characteristics of lysine side chain, such as flexibility, polarity, and the energetic cost upon burial, suggest that this lysine is one of the structural designs in regulating metastability and function of inhibitory serpins in general.

The unfolding enthalpy of the pH 4 molten globule from sperm whale apomyoglobin has been measured by isothermal titration calorimetry, using titration to acid pH. The unfolding enthalpy is close to zero at 20 °C, in contrast both to the positive values expected for peptide helices and the negative values reported for holomyoglobin and native apomyoglobin. At 20 °C, the hydrophobic interaction should make only a small contribution to the unfolding enthalpy according to the liquid hydrocarbon model. Our result indicates that some factor present in the unfolding enthalpies of native proteins makes the unfolding enthalpy of the pH 4 molten globule less positive than expected from data for peptide helices.

To provide a framework for understanding the hyperthermostability of some rubredoxins, a comprehensive analysis of the thermally induced denaturation of rubredoxin (Rd) from the mesophile, Clostridium pasteurianum was undertaken. Rds with three different metals in its M(SCys)4 site (M = Fe3+/2+, Zn2+, or Cd2+) were examined. Kinetics of metal ion release were monitored anaerobically at several fixed temperatures between 40 and 100 °C, and during progressive heating of the iron-containing protein. Both methods gave a thermal stability of metal binding in the order Fe2+ [Lt ] Fe3+ < Zn2+ < Cd2+. The temperature at which half of the iron was released from the protein in temperature ramp experiments was 69 °C for Fe2+Rd and 83 °C for Fe3+Rd. Temperature-dependent changes in the protein structure were monitored by differential scanning calorimetry, tryptophan fluorescence, binding of a fluorescent hydrophobic probe, and 1H NMR. Major but reversible structural changes, consisting of swelling of the hydrophobic core and opening of a loop region, were found to occur at temperatures (50–70 °C) much lower than those required for loss of the metal ion. For the three divalent metal ions, the results suggest that the onset of the reversible, lower-temperature structural changes is dependent on the size of the MS4 site, whereas the final, irreversible loss of metal ion is dependent on the inherent M-SCys bond strength. In the case of Fe3+Rd, stoichiometric Fe3+/cysteine–ligand redox chemistry also occurs during metal ion loss. The results indicate that thermally induced unfolding of the native Cp Rd must surmount a significant kinetic barrier caused by stabilizing interactions both within the protein and within the M(SCys)4 site.

The heat stable inhibitor of cAMP-dependent protein kinase (PKIα) contains both a nuclear export signal (NES) and a high affinity inhibitory region that is essential for inhibition of the catalytic subunit of the kinase. These functions are sequentially independent. Two-dimensional NMR spectroscopy was performed on uniformly [15N]-labeled PKIα to examine its structure free in solution. Seventy out of 75 residues were identified, and examination of the CαH chemical shifts revealed two regions of upfield chemical shifts characteristic of α-helices. When PKIα was fragmented into two functionally distinct peptides for study at higher concentrations, no significant alterations in chemical shifts or secondary structure were observed. The first ordered region, identified in PKIα (1–25), contains an α-helix from residues 1–13. This helix extends by one turn the helix observed in the crystal structure of a PKIα (5–24) peptide bound to the catalytic subunit. The second region of well-defined secondary structure, residues 35–47, overlaps with the nuclear export signal in the PKIα (26–75) fragment. This secondary structure consists of a helix with a hydrophobic face comprised of Leu37, Leu41, and Leu44, followed by a flexible turn containing Ile46. These four residues are critical for nuclear export function. The remainder of the protein in solution appears relatively unstructured, and this lack of structure surrounding a few essential and well-defined signaling elements may be characteristic of a growing family of small regulatory proteins that interact with protein kinases.

Maltose binding protein (MBP) is a large, monomeric two domain protein containing 370 amino acids. In the absence of denaturant at neutral pH, the protein is in the native state, while at pH 3.0 it forms a molten globule. The molten globule lacks a tertiary circular dichroism signal but has secondary structure similar to that of the native state. The molten globule binds 8-anilino-1-naphthalene sulfonate (ANS). The unfolding thermodynamics of MBP at both pHs were measured by carrying out a series of isothermal urea melts at temperatures ranging from 274–329 K. At 298 K, values of ΔG°, ΔCp, and Cm were 3.1 ± 0.2 kcal mol−1, 5.9 ± 0.8 kcal mol−1 K−1 (15.9 cal (mol-residue)−1 K−1), and 0.8 M, respectively, at pH 3.0 and 14.5 ± 0.4 kcal mol−1, 8.3 ± 0.7 kcal mol−1 K−1 (22.4 kcal (mol-residue)−1 K−1), and 3.3 M, respectively, at pH 7.1. Guanidine hydrochloride denaturation at pH 7.1 gave values of ΔG° and ΔCp similar to those obtained with urea. The m values for denaturation are strongly temperature dependent, in contrast to what has been previously observed for small globular proteins. The value of ΔCp per mol-residue for the molten globule is comparable to corresponding values of ΔCp for the unfolding of typical globular proteins and suggests that it is a highly ordered structure, unlike molten globules of many small proteins. The value of ΔCp per mol-residue for the unfolding of the native state is among the highest currently known for any protein.

The stability of protein is defined not only by the hydrogen bonding, hydrophobic effect, van der Waals interactions, and salt bridges. Additional, much more subtle contributions to protein stability can arise from surface residues that change their properties upon unfolding. The recombinant major cold shock protein of Escherichia coli CspA an all-β protein unfolds reversible in a two-state manner, and behaves in all other respects as typical globular protein. However, the enthalpy of CspA unfolding strongly depends on the pH and buffer composition. Detailed analysis of the unfolding enthalpies as a function of pH and buffers with different heats of ionization shows that CspA unfolding in the pH range 5.5–9.0 is linked to protonation of an amino group. This amino group appears to be the N-terminal α-amino group of the CspA molecule. It undergoes a 1.6 U shift in pKa values between native and unfolded states. Although this shift in pKa is expected to contribute ∼5 kJ/mol to CspA stabilization energy the experimentally observed stabilization is only ∼1 kJ/mol. This discrepancy is related to a strong enthalpy–entropy compensation due, most likely, to the differences in hydration of the protonated and deprotonated forms of the α-amino group.

Endo-β-N-acetylglucosaminidase H hydrolyzes the β-(1–4)-glycosidic link of the N,N′-diacetylchitobiose core of high-mannose and hybrid asparagine-linked oligosaccharides. Seven mutants of the active site residues, Asp130 and Glu132, have been prepared, assayed, and crystallized. They include single site mutants of each residue to the corresponding amide, to Ala and to the alternate acidic residue, and to the double amide mutant. The mutants of Asp130 are more active than the corresponding Glu132 mutants, consistent with the assignment of the latter residue as the primary catalytic residue. The amide mutants are more active than the alternate acidic residue mutants, which in turn are more active than the Ala mutants. The structures of the Asn mutant of Asp130 and the double mutant are very similar to that of the wild-type enzyme. Several residues surrounding the mutated residues, including some that form part of the core of the β-barrel and especially Tyr168 and Tyr244, adopt a very different conformation in the structures of the other two mutants of Asp130 and in the Asp mutant of Glu132. The results show that the residues in the upper layers of the β-barrel can organize into two very distinct packing arrangements that depend on subtle electrostatic and steric differences and that greatly affect the geometry of the substrate-binding cleft. Consequently, the relative activities of several of the mutants are defined by structural changes, leading to impaired substrate binding, in addition to changes in functionality.

The 2.5 Å crystal structure of the full length human placental isoform of the Gly12 to Val mutant Cdc42 protein (Cdc42(G12V)) bound to both GDP/Mg2+ and GDPNH2 (guanosine-5′-diphospho-β-amidate) is reported. The crystal contains two molecules in the asymmetric unit, of which one has bound GDP/Mg2+, while the other has bound GDPNH2 without a Mg2+ ion. Crystallization of the protein was induced via hydrolysis of the Cdc42·GppNHp complex by the presence of contaminating alkaline phosphatase activity in combination with the crystallization conditions. This prompted us to compare the binding characteristics of GDPNH2 vs. GDP. The amino group of GDPNH2 drastically reduces the affinity to Cdc42 in comparison with that of GDP, causes the loss of the Mg2+ ion, and apparently also increases the conformational flexibility of the protein as seen in the crystal. Both the switch I and switch II regions are visible in the electron density of the GDP-bound molecule, but not in the molecule bound to GDPNH2. The C-terminus containing the CaaX-motif is partly ordered in both molecules due to an intramolecular disulfide bond formed between Cys105/Cys188 and Cys305/Cys388, respectively.

Molecular dynamics calculations provide a method by which the dynamic properties of molecules can be explored over timescales and at a level of detail that cannot be obtained experimentally from NMR or X-ray analyses. Recent work (Philippopoulos M, Mandel AM, Palmer AG III, Lim C, 1997, Proteins 28:481–493) has indicated that the accuracy of these simulations is high, as measured by the correspondence of parameters extracted from these calculations to those determined through experimental means. Here, we investigate the dynamic behavior of the Src homology 3 (SH3) domain of hematopoietic cell kinase (Hck) via 15N backbone relaxation NMR studies and a set of four independent 4 ns solvated molecular dynamics calculations. We also find that molecular dynamics simulations accurately reproduce fast motion dynamics as estimated from generalized order parameter (S2) analysis for regions of the protein that have experimentally well-defined coordinates (i.e., stable secondary structural elements). However, for regions where the coordinates are not well defined, as indicated by high local root-mean-square deviations among NMR-determined structural family members or high B-factors/low electron density in X-ray crystallography determined structures, the parameters calculated from a short to moderate length (less than 5–10 ns) molecular dynamics trajectory are dependent on the particular coordinates chosen as a starting point for the simulation.

The first crystal structure of an inorganic pyrophosphatase (S-PPase) from an archaebacterium, the thermophile Sulfolobus acidocaldarius, has been solved by molecular replacement and refined to an R-factor of 19.7% at 2.7 Å. S-PPase is a D3 homohexameric protein with one Mg2+ per active site in a position similar to, but not identical with, the first activating metal in mesophilic pyrophosphatases (PPase). In mesophilic PPases, Asp65, Asp70, and Asp102 coordinate the Mg2+, while only Asp65 and Asp102 do in S-PPase, and the Mg2+ moves by 0.7 Å. S-PPase may therefore be deactivated at low temperature by mispositioning a key metal ion.

The monomer S-PPase structure is very similar to that of Thermus thermophilus (T-PPase) and Escherichia coli (E-PPase), root-mean-square deviations around 1 Å/Cα. But the hexamer structures of S- and T-PPase are more tightly packed and more similar to each other than they are to that of E-PPase, as shown by the increase in surface area buried upon oligomerization. In T-PPase, Arg116 creates an interlocking ionic network to both twofold and threefold related monomers; S-PPase has hydrophilic interactions to threefold related monomers absent in both E- and T-PPase. In addition, the thermostable PPases have about 7% more hydrogen bonds per monomer than E-PPase, and, especially in S-PPase, additional ionic interactions anchor the C-terminus to the rest of the protein. Thermostability in PPases is thus due to subtle improvements in both monomer and oligomer interactions.

Comparison of the inferred amino acid sequence of orf AF1736 of Archaeoglobus fulgidus to that of Pseudomonas mevalonii HMG-CoA reductase suggested that AF1736 might encode a Class II HMG-CoA reductase. Following polymerase chain reaction–based cloning of AF1736 from A. fulgidus genomic DNA and expression in Escherichia coli, the encoded enzyme was purified to apparent homogeneity and its enzymic properties were determined. Activity was optimal at 85 °C, ΔHa was 54 kJ/mol, and the statin drug mevinolin inhibited competitively with HMG-CoA (Ki 180 μM). Protonated forms of His390 and Lys277, the apparent cognates of the active site histidine and lysine of the P. mevalonii enzyme, appear essential for activity. The mechanism proposed for catalysis of P. mevalonii HMG-CoA reductase thus appears valid for A. fulgidus HMG-CoA reductase. Unlike any other HMG-CoA reductase, the A. fulgidus enzyme exhibits dual coenzyme specificity. pH-activity profiles for all four reactions revealed that optimal activity using NADP(H) occurred at a pH from 1 to 3 units more acidic than that observed using NAD(H). Kinetic parameters were therefore determined for all substrates for all four catalyzed reactions using either NAD(H) or NADP(H). NADPH and NADH compete for occupancy of a common site. kcat[NAD(H)]/kcat[NADP(H)] varied from unity to under 70 for the four reactions, indicative of slight preference for NAD(H). The results indicate the importance of the protonated status of active site residues His390 and Lys277, shown by altered KM and kcat values, and indicate that NAD(H) and NADP(H) have comparable affinity for the same site.

The extracellular portions of the chains that comprise the human type I interferon receptor, IFNAR1 and IFNAR2, have been expressed and purified as recombinant soluble His-tagged proteins, and their interactions with each other and with human interferon-β-1a (IFN-β-1a) were studied by gel filtration and by cross-linking. By gel filtration, no stable binary complexes between IFN-β-1a and IFNAR1, or between IFNAR1 and IFNAR2 were detected. However, a stable binary complex formed between IFN-β-1a and IFNAR2. Analysis of binary complex formation using various molar excesses of IFN-β-1a and IFNAR2 indicated that the complex had a 1:1 stoichiometry, and reducing SDS-PAGE of the binary complex treated with the cross-linking reagent dissucinimidyl glutarate (DSG) indicated that the major cross-linked species had an apparent M>r consistent with the sum of its two individual components. Gel filtration of a mixture of IFNAR1 and the IFN-β-1a/IFNAR2 complex indicated that the three proteins formed a stable ternary complex. Analysis of ternary complex formation using various molar excesses of IFNAR1 and the IFN-β-1a/IFNAR2 complex indicated that the ternary complex had a 1:1:1 stoichiometry, and reducing SDS-PAGE of the ternary complex treated with DSG indicated that the major cross-linked species had an apparent Mr consistent with the sum of its three individual components. We conclude that the ternary complex forms by the sequential association of IFN-β-1a with IFNAR2, followed by the association of IFNAR1 with the preformed binary complex. The ability to produce the IFN-β-1a/IFNAR2 and IFN-β-1a/IFNAR1/IFNAR2 complexes make them attractive candidates for X-ray crystallography studies aimed at determining the molecular interactions between IFN-β-1a and its receptor.

To explore the structure of the pore-forming fragment of colicin E1 in membranes, a series of 23 consecutive single cysteine substitution mutants was prepared in the sequence 402–424. Each mutant was reacted with a sulfhydryl-specific reagent to generate a nitroxide labeled side chain, and the mobility of the side chain and its accessibility to collision with paramagnetic reagents was determined from the electron paramagnetic resonance spectrum. Individual values of these quantities were used to identify tertiary contact sites and the nature of the surrounding solvent, while their periodic dependence on sequence position was used to identify secondary structure. In solution, the data revealed a regular helix of 11 residues in the region 406–416, consistent with helix IV of the crystal structure. Upon binding to negatively charged membranes at pH 4.0, helix IV apparently grows to a length of 19 residues, extending from 402–420. One face of the helix is solvated by the lipid bilayer, and the other by an environment of a polar nature. Surprisingly, a conserved charged pair, D408–R409, is located on the lipid-exposed face. Evidence is presented to suggest a transmembrane orientation of this new helix, although other topographies may exist in equilibrium.

The structure and dynamics of the fatty acid binding cavity in I-FABP (rat intestinal fatty acid binding protein) were analyzed. In the crystal structure of apo I-FABP, the probe occupied cavity volume and surface are 539 ± 8 Å3 and 428 Å2, respectively (1.4 Å probe). A total of 31 residues contact the cavity with their side chains. The side-chain cavity surface is partitioned according to the residue type as follows: 36–39% hydrophobic, 21–25% hydrophilic, and 37–43% neutral or ambivalent. Thus, the cavity surface is neither like a typical protein interior core, nor is like a typical protein external surface. All hydrophilic residues that contact the cavity—with the exception of Asp74—are clustered on the one side of the cavity. The cavity appears to expand its hydrophobic surface upon fatty acid binding on the side opposite to this hydrophilic patch. In holo I-FABP the fatty acid chain interactions with the hydrophilic side chains are mediated by water molecules. Molecular dynamics (MD) simulation of fully solvated apo I-FABP showed global conformational changes of I-FABP, which resulted in a large, but seemingly transient, exposure of the cavity to the external solvent. The packing density of the side chains lining the cavity, studied by Voronoi volumes, showed the presence of two distinctive small hydrophobic cores. The MD simulation predicts significant structural perturbations of the cavity on the subnanosecond time scale, which are capable of facilitating exchange of I-FABP internal water.