Protein Science

The X‐ray crystal structure of human transglutaminase factor XIII has revealed a cysteine proteinase‐like active site involved in a crosslinking reaction and not proteolysis. This is among the first observations of similar active sites in 2 different enzyme families catalyzing a similar reaction in opposite directions. Although the size and overall protein fold of factor XIII and the cysteine proteinases are quite different, the active site and the surrounding protein structure share structural features suggesting a common evolutionary lineage. Here we present a description of the residues in the active site and the structural evidence that the catalytic mechanism of the transglutaminases is similar to the reverse mechanism of the cysteine proteinases.

Polyproline II (PPII) is reported to be a dominant conformation in the unfolded state of peptides, even when no prolines are present in the sequence. Here we use isothermal titration calorimetry (ITC) to investigate the PPII bias in the unfolded state by studying the binding of the SH3 domain of SEM‐5 to variants of its putative PPII peptide ligand, Sos. The experimental system is unique in that it provides direct access to the conformational entropy change of the substituted amino acids. Results indicate that the denatured ensemble can be characterized by at least two thermodynamically distinct states, the PPII conformation and an unfolded state conforming to the previously held idea of the denatured state as a random collection of conformations determined largely by hard‐sphere collision. The probability of the PPII conformation in the denatured states for Ala and Gly were found to be significant, ∼30% and ∼10%, respectively, resulting in a dramatic reduction in the conformational entropy of folding.

We have identified a novel, conserved phosphatase sequence motif, KXXXXXXRP‐(X_12‐54)‐PSGH‐(X_31‐54))‐SRXXXXX HXXXD, that is shared among several lipid phosphatases, the mammalian glucose‐6‐phosphatases, and a collection of bacterial nonspecific acid phosphatases. This sequence was also found in the vanadium‐containing chloroperoxidase of Curvularia inaequalis. Several lines of evidence support this phosphatase motif identification. Crystal structure data on chloroperoxidase revealed that all three domains are in close proximity and several of the conserved residues are involved in the binding of the cofactor, vana‐date, a compound structurally similar to phosphate. Structure‐function analysis of the human glucose‐6‐phosphatase has shown that two of the conserved residues (the first domain arginine and the central domain histidine) are essential for enzyme activity. This conserved sequence motif was used to identify nine additional putative phosphatases from sequence databases, one of which has been determined to be a lipid phosphatase in yeast.

The structures of the oxidized and reduced forms of the rubredoxin from the archaebacterium, Pyrococcus furiosus, an organism that grows optimally at 100 °C, have been determined by X‐ray crystallography to a resolution of 1.8 å. Crystals of this rubredoxin grow in space group P2₁2₁2₁ with room temperature cell dimensions a = 34.6 å, b = 35.5 å, and c = 44.4 å. Initial phases were determined by the method of molecular replacement using the oxidized form of the rubredoxin from the mesophilic eubacterium, Clostridium pasteurianum, as a starting model. The oxidized and reduced models of P. furiosus rubredoxin each contain 414 nonhydrogen protein atoms comprising 53 residues. The model of the oxidized form contains 61 solvent H₂O oxygen atoms and has been refined with X‐PLOR and TNT to a final R = 0.178 with root mean square (rms) deviations from ideality in bond distances and bond angles of 0.014 å and 2.06°, respectively. The model of the reduced form contains 37 solvent H₂O oxygen atoms and has been refined to R = 0.193 with rms deviations from ideality in bond lengths of 0.012 å and in bond angles of 1.95°. The overall structure of P. furiosus rubredoxin is similar to the structures of mesophilic rubredoxins, with the exception of a more extensive hydrogen‐bonding network in the β‐sheet region and multiple electrostatic interactions (salt bridge, hydrogen bonds) of the Glu 14 side chain with groups on three other residues (the amino‐terminal nitrogen of Ala 1; the indole nitrogen of Trp 3; and the amide nitrogen group of Phe 29). The influence of these and other features upon the thermostability of the P. furiosus protein is discussed.

We apply a simple method for aligning protein sequences on the basis of a 3D structure, on a large scale, to the proteins in the scop classification of fold families. This allows us to assess, understand, and improve our automatic method against an objective, manually derived standard, a type of comprehensive evaluation that has not yet been possible for other structural alignment algorithms. Our basic approach directly matches the backbones of two structures, using repeated cycles of dynamic programming and least‐squares fitting to determine an alignment minimizing coordinate difference. Because of simplicity, our method can be readily modified to take into account additional features of protein structure such as the orientation of side chains or the location‐dependent cost of opening a gap. Our basic method, augmented by such modifications, can find reasonable alignments for all but 1.5% of the known structural similarities in scop, i.e., all but 32 of the 2,107 superfamily pairs. We discuss the specific protein structural features that make these 32 pairs so difficult to align and show how our procedure effectively partitions the relationships in scop into different categories, depending on what aspects of protein structure are involved (e.g., depending on whether or not consideration of side‐chain orientation is necessary for proper alignment). We also show how our pairwise alignment procedure can be extended to generate a multiple alignment for a group of related structures. We have compared these alignments in detail with corresponding manual ones culled from the literature. We find good agreement (to within 95% for the core regions), and detailed comparison highlights how particular protein structural features (such as certain strands) are problematical to align, giving somewhat ambiguous results. With these improvements and systematic tests, our procedure should be useful for the development of scop and the future classification of protein folds. Supplementary material is available at http://bioinfo.mbb.yale.edu/align.

Protein structures in the Protein Data Bank provide a wealth of data about the interactions that determine the native states of proteins. Using the probability theory, we derive an atomic distance‐dependent statistical potential from a sample of native structures that does not depend on any adjustable parameters (Discrete Optimized Protein Energy, or DOPE). DOPE is based on an improved reference state that corresponds to noninteracting atoms in a homogeneous sphere with the radius dependent on a sample native structure; it thus accounts for the finite and spherical shape of the native structures. The DOPE potential was extracted from a nonredundant set of 1472 crystallographic structures. We tested DOPE and five other scoring functions by the detection of the native state among six multiple target decoy sets, the correlation between the score and model error, and the identification of the most accurate non‐native structure in the decoy set. For all decoy sets, DOPE is the best performing function in terms of all criteria, except for a tie in one criterion for one decoy set. To facilitate its use in various applications, such as model assessment, loop modeling, and fitting into cryo‐electron microscopy mass density maps combined with comparative protein structure modeling, DOPE was incorporated into the modeling package MODELLER‐8.

The structure of phthalate dioxygenase reductase (PDR), a monomeric iron‐sulfur flavoprotein that delivers electrons from NADH to phthalate dioxygenase, is compared to ferredoxin‐NADP⁺ reductase (FNR) and ferredoxin, the proteins that reduce NADP⁺ in the final reaction of photosystem I. The folding patterns of the domains that bind flavin, NAD(P), and [2Fe‐2S] are very similar in the two systems. Alignment of the X‐ray structures of PDR and FNR substantiates the assignment of features that characterize a family of flavoprotein reductases whose members include cytochrome P‐450 reductase, sulfite and nitrate reductases, and nitric oxide synthase. Hallmarks of this subfamily of flavoproteins, here termed the FNR family, are an antiparallel β‐barrel that binds the flavin prosthetic group, and a characteristic variant of the classic pyridine nucleotide‐binding fold. Despite the similarities between FNR and PDR, attempts to model the structure of a dissociable FNR:ferredoxin complex by analogy with PDR reveal features that are at odds with chemical crosslinking studies (Zanetti, G., Morelli, D., Ronchi, S., Negri, A., Aliverti, A., & Curti, B., 1988, Biochemistry 27, 3753–3759).

Differences in the binding sites for flavin and pyridine nucleotides determine the nucleotide specificities of FNR and PDR. The specificity of FNR for NADP⁺ arises primarily from substitutions in FNR that favor interactions with the 2′ phosphate of NADP⁺. Variations in the conformation and sequences of the loop adjoining the flavin phosphate affect the selectivity for FAD versus FMN.

The midpoint potentials for reduction of the flavin and [2Fe–2S] groups in PDR are higher than their counterparts in FNR and spinach ferredoxin, by about 120 mV and 260 mV, respectively. Comparisons of the structure of PDR with spinach FNR and with ferredoxin from Anabaena 7120, along with calculations of electrostatic potentials, suggest that local interactions, including hydrogen bonds, are the dominant contributors to these differences in potential.

Glutathione S‐transferases (GSTs) are dimeric proteins that play an important role in cellular detoxification. Four GSTs from the mosquito Anopheles dirus species B (Ad), an important malaria vector in South East Asia, are produced by alternate splicing of a single transcription product and were previously shown to have detoxifying activity towards pesticides such as DDT. We have determined the crystal structures for two of these alternatively spliced proteins, AdGST1–3 (complexed with glutathione) and AdGST1–4 (apo form), at 1.75 and 2.45 Å resolution, respectively. These GST isozymes show differences from the related GST from the Australian sheep blowfly Lucilia cuprina; in particular, the presence of a C‐terminal helix forming part of the active site. This helix causes the active site of the Anopheles GSTs to be enclosed. The glutathione‐binding helix α2 and flanking residues are disordered in the AdGST1–4 (apo) structure, yet ordered in the AdGST1–3 (GSH‐bound) structure, suggesting that insect GSTs operate with an induced fit mechanism similar to that found in the plant phi‐ and human pi‐class GSTs. Despite the high overall sequence identities, the active site residues of AdGST1–4 and AdGST1–3 have different conformations.

We studied the non‐native aggregation of recombinant human granulocyte stimulating factor (rhGCSF) in solution conditions where native rhGCSF is both conformationally stable compared to its unfolded state and at concentrations well below its solubility limit. Aggregation of rhGCSF first involves the perturbation of its native structure to form a structurally expanded transition state, followed by assembly process to form an irreversible aggregate. The energy barriers of the two steps are reflected in the experimentally measured values of free energy of unfolding (ΔG_unf) and osmotic second virial coefficient (B₂₂), respectively. Under solution conditions where rhGCSF conformational stability dominates (i.e., large ΔG_unf and negative B₂₂), the first step is rate‐limiting, and increasing ΔG_unf (e.g., by the addition of sucrose) decreases aggregation. In solutions where colloidal stability is high (i.e., large and positive B₂₂ values) the second step is rate‐limiting, and solution conditions (e.g., low pH and low ionic strength) that increase repulsive interactions between protein molecules are effective at reducing aggregation. rhGCSF aggregation is thus controlled by both conformational stability and colloidal stability, and depending on the solution conditions, either could be rate‐limiting.