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Unfolded and partially unfolded proteins participate in a wide range of biological processes from pathological aggregation to the regulation of normal cellular activity. Unfolded states can be populated under strongly denaturing conditions, but the ensemble which is relevant for folding, stability, and aggregation is that populated under physiological conditions. Characterization of nonnative states is critical for the understanding of these processes, yet comparatively little is known about their energetics and their structural propensities under native conditions. The standard view is that energetically significant coupled interactions involving multiple residues are generally not present in the denatured state ensemble (DSE) or in intrinsically disordered proteins. Using the N-terminal domain of the ribosomal protein L9, a small α-β protein, as an experimental model system, we demonstrate that networks of energetically significant, coupled interactions can form in the DSE of globular proteins, and can involve residues that are distant in sequence and spatially well separated in the native structure. X-ray crystallography, NMR, dynamics studies, native state pKa measurements, and thermodynamic analysis of more than 25 mutants demonstrate that residues are energetically coupled in the DSE. Altering these interactions by mutation affects the stability of the domain. Mutations that alter the energetics of the DSE can impact the analysis of cooperativity and folding, and may play a role in determining the propensity to aggregate.

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2008.05.046 Byline: Imsang Lee (1), Hermann Schindelin (1)(2) Keywords: PROTEINS Abstract: Ubiquitin (Ub) and ubiquitin-like proteins (Ubls) are conjugated to their targets by specific cascades involving three classes of enzymes, E1, E2, and E3. Each E1 adenylates the C terminus of its cognate Ubl, forms a E1[approximately equal to]Ubl thioester intermediate, and ultimately generates a thioester-linked E2[approximately equal to]Ubl product. We have determined the crystal structure of yeast Uba1, revealing a modular architecture with individual domains primarily mediating these specific activities. The negatively charged C-terminal ubiquitin-fold domain (UFD) is primed for binding of E2s and recognizes their positively charged first [alpha] helix via electrostatic interactions. In addition, a mobile loop from the domain harboring the E1 catalytic cysteine contributes to E2 binding. Significant, experimentally observed motions in the UFD around a hinge in the linker connecting this domain to the rest of the enzyme suggest a conformation-dependent mechanism for the transthioesterification function of Uba1; however, this mechanism clearly differs from that of other E1 enzymes. Author Affiliation: (1) Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY 11794-5215, USA (2) Rudolf Virchow Center for Experimental Biomedicine and Institute for Structural Biology, University of Wurzburg, Versbacher Str. 9, 97078 Wurzburg, Germany Article History: Received 9 October 2007; Revised 20 March 2008; Accepted 12 May 2008 Article Note: (miscellaneous) Published: July 24, 2008

The crystal structures of non-thiocarboxylated molybdopterin (MPT) synthase from Staphylococcus aureus in its apo form and in complex with precursor Z are presented. A comparison of the structures reveals conformational changes in a loop that participates in interactions with precursor Z, with biochemical evidence revealing its role in sulphur transfer mechanism and molybdenum cofactor deficiency.

A detailed mutational analysis of the MoeA protein from Escherichia coli, which mediates ligation of Mo to molybdopterin, is described. Crystal structures of the functionally characterized MoeA variants revealed no major structural changes, indicating that the functional differences observed are not due to disruption of the protein structure.

Phosphotransacetylase (EC 2.3.1.8) catalyzes reversible transfer of the acetyl group from acetyl phosphate to coenzyme A (CoA), forming acetyl-CoA and inorganic phosphate. Two crystal structures of phosphotransacetylase from the methanogenic archaeon Methanosarcina thermophila in complex with the substrate CoA revealed one CoA (Co[A.sup.l]) bound in the proposed active site cleft and an additional CoA (Co[A.sup.2]) bound at the periphery of the cleft. The results of isothermal titration calorimetry experiments are described, and they support the hypothesis that there are distinct high-affinity (equilibrium dissociation constant [[K.sub.D]], 20 [micro]M) and low-affinity ([K.sub.D], 2 mM) CoA binding sites. The crystal structures indicated that binding of Co[A.sub.1] is mediated by a series of hydrogen bonds and extensive van der Waals interactions with the enzyme and that there are fewer of these interactions between Co[A.sup.2] and the enzyme. Different conformations of the protein observed in the crystal structures suggest that domain movements which alter the geometry of the active site cleft may contribute to catalysis. Kinetic and calorimetric analyses of site-specific replacement variants indicated that there are catalytic roles for [Ser.sup.309] and [Arg.sup.310], which are proximal to the reactive sulfhydryl of Co[A.sup.1]. The reaction is hypothesized to proceed through base-catalyzed abstraction of the thiol proton of CoA by the adjacent and invariant residue [Asp.sup.316], followed by nucleophilic attack of the thiolate anion of CoA on the carbonyl carbon of acetyl phosphate. We propose that [Arg.sup.310] binds acetyl phosphate and orients it for optimal nucleophilic attack. The hypothesized mechanism proceeds through a negatively charged transition state stabilized by hydrogen bond donation from [Ser.sup.309].

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2005.10.044 Byline: Geng Tian (1)(2), Song Xiang (2), Robert Noiva (3), William J. Lennarz (1), Hermann Schindelin (1)(2)(4) Abstract: Protein disulfide isomerase plays a key role in catalyzing the folding of secretory proteins. It features two catalytically inactive thioredoxin domains inserted between two catalytically active thioredoxin domains and an acidic C-terminal tail. The crystal structure of yeast PDI reveals that the four thioredoxin domains are arranged in the shape of a twisted 'U' with the active sites facing each other across the long sides of the 'U.' The inside surface of the 'U' is enriched in hydrophobic residues, thereby facilitating interactions with misfolded proteins. The domain arrangement, active site location, and surface features strikingly resemble the Escherichia coli DsbC and DsbG protein disulfide isomerases. Biochemical studies demonstrate that all domains of PDI, including the C-terminal tail, are required for full catalytic activity. The structure defines a framework for rationalizing the differences between the two active sites and their respective roles in catalyzing the formation and rearrangement of disulfide bonds. Author Affiliation: (1) Department of Biochemistry and Cell Biology, State University of New York at Stony Brook, Stony Brook, NY 11794, USA (2) Center for Structural Biology, State University of New York at Stony Brook, Stony Brook, NY 11794, USA (3) Division of Basic Biomedical Sciences, University of South Dakota, School of Medicine, Vermillion, SD 57069, USA (4) Rudolf Virchow Center for Experimental Biomedicine and Institute of Structural Biology, University of Wurzburg, Versbacher Str. 9, 97078 Wurzburg, Germany Article History: Received 16 June 2005; Revised 24 August 2005; Accepted 11 October 2005 Article Note: (miscellaneous) Published: January 12, 2006

The crystal structure of yeast protein disulfide isomerase reveals that the four thioredoxin domains are arranged in the shape of a twisted 'U' with the active sites facing each other across the long sides of the 'U'. The structure defines a framework for rationalizing the differences between the two active sites and their respective roles in catalyzing the formation and rearrangement of disulfide bonds.

Author(s): Michael W. Lake [1]; Margot M. Wuebbens [2]; K. V. Rajagopalan [2]; Hermann Schindelin (corresponding author) [1] The activation of ubiquitin and related protein modifiers [1, 2] is catalysed [...]