Structure (v.13, #2)

SAXS and the Working Protein by Bhushan Nagar; John Kuriyan (169-170).
In this issue of Structure, Davies et al., 2005 present shape reconstructions for the molecular motor p97 using small angle X-ray scattering (SAXS) and offer insights into how ATP consumption is coupled to cyclical domain motions. This work emphasizes the emerging potential of SAXS for visualizing the workings of biological machines in solution.

Low-Resolution Crystallography Is Coming of Age by Axel T. Brunger (171-172).
The threshold of what is considered “acceptable” resolution for obtaining mechanistic insights is being pushed by recent structures at 3.8 to 4.7 Å resolution. One of these structures, that of a fully glycosylated SIV gp120 envelope glycoprotein in an unliganded conformation at 4.0 Å resolution, is described in this issue ().

Dynein Cargo Gets Its Groove Back by K. Kevin Pfister (172-173).
Recent work from the King lab () on the structure of the Tctex1 dynein light chain provides new insights into the mechanism of cytoplasmic dynein cargo binding and the functional significance of light chain isoform diversity.

Benefits of Automated Crystallization Plate Tracking, Imaging, and Analysis by Christopher J. Mayo; Jonathan M. Diprose; Thomas S. Walter; Ian M. Berry; Julie Wilson; Ray J. Owens; E. Yvonne Jones; Karl Harlos; David I. Stuart; Robert M. Esnouf (175-182).
We describe the design of a database and software for managing and organizing protein crystallization data. We also outline the considerations behind the design of a fast web interface linking protein production data, crystallization images, and automated image analysis. The database and associated interfaces underpin the Oxford Protein Production Facility (OPPF) crystallization laboratory, collecting, in a routine and automatic manner, up to 100,000 images per day. Over 17 million separate images are currently held in this database. We discuss the substantial scientific benefits automated tracking, imaging, and analysis of crystallizations offers to the structural biologist: analysis of the time course of the trial and easy analysis of trials with related crystallization conditions. Features of this system address requirements common to many crystallographic laboratories that are currently setting up (semi-)automated crystallization imaging systems.

Conformational Changes of p97 during Nucleotide Hydrolysis Determined by Small-Angle X-Ray Scattering by Jason M. Davies; Hirotsugu Tsuruta; Andrew P. May; William I. Weis (183-195).
Valosin-containing protein (VCP)/p97 is an AAA family ATPase that has been implicated in the removal of misfolded proteins from the endoplasmic reticulum and in membrane fusion. p97 forms a homohexamer whose protomers consist of an N-terminal (N) domain responsible for binding to effector proteins, followed by two AAA ATPase domains, D1 and D2. Small-angle X-ray scattering (SAXS) measurements of p97 in the presence of AMP-PNP (ATP state), ADP-AlFx (hydrolysis transition state), ADP, or no nucleotide reveal major changes in the positions of the N domains with respect to the hexameric ring during the ATP hydrolysis cycle. Nucleotide binding and hydrolysis experiments indicate that D2 inhibits nucleotide exchange by D1. The data suggest that the conversion of the chemical energy of ATP hydrolysis into mechanical work on substrates involves transmission of conformational changes generated by D2 through D1 to move N.

Determining the Structure of an Unliganded and Fully Glycosylated SIV gp120 Envelope Glycoprotein by Bing Chen; Erik M. Vogan; Haiyun Gong; John J. Skehel; Don C. Wiley; Stephen C. Harrison (197-211).
HIV/SIV envelope glycoproteins mediate the first steps in viral infection. They are trimers of a membrane-anchored polypeptide chain, cleaved into two fragments known as gp120 and gp41. The structure of HIV gp120 bound with receptor (CD4) has been known for some time. We have now determined the structure of a fully glycosylated SIV gp120 envelope glycoprotein in an unliganded conformation by X-ray crystallography at 4.0 Å resolution. We describe here our experimental and computational approaches, which may be relevant to other resolution-limited crystallographic problems. Key issues were attention to details of beam geometry mandated by small, weakly diffracting crystals, and choice of strategies for phase improvement, starting with two isomorphous derivatives and including multicrystal averaging. We validated the structure by analyzing composite omit maps, averaged among three distinct crystal lattices, and by calculating model-based, SeMet anomalous difference maps. There are at least four ordered sugars on many of the thirteen oligosaccharides.

Solution Structure of the Tctex1 Dimer Reveals a Mechanism for Dynein-Cargo Interactions by Hongwei Wu; Mark W. Maciejewski; Sachiko Takebe; Stephen M. King (213-223).
Tctex1 is a light chain found in both cytoplasmic and flagellar dyneins and is involved in many fundamental cellular activities, including rhodopsin transport within photoreceptors, and may function in the non-Mendelian transmission of t haplotypes in mice. Here, we present the NMR solution structure for the Tctex1 dimer from Chlamydomonas axonemal inner dynein arm I1. Structural comparisons reveal a strong similarity with the LC8 dynein light chain dimer, including formation of a strand-switched β sheet interface. Analysis of the Tctex1 structure enables the dynein intermediate chain binding site to be identified and suggests a mechanism by which cargo proteins might be attached to this microtubule motor complex. Comparison with the alternate dynein light chain rp3 reveals how the specificity of dynein-cargo interactions mediated by these dynein components is achieved. In addition, this structure provides insight into the consequences of the mutations found in the t haplotype forms of this protein.

Design of a Heterospecific, Tetrameric, 21-Residue Miniprotein with Mixed α/β Structure by Mayssam H. Ali; Christina M. Taylor; Gevorg Grigoryan; Karen N. Allen; Barbara Imperiali; Amy E. Keating (225-234).
The study of short, autonomously folding peptides, or “miniproteins,” is important for advancing our understanding of protein stability and folding specificity. Although many examples of synthetic α-helical structures are known, relatively few mixed α/β structures have been successfully designed. Only one mixed-secondary structure oligomer, an α/β homotetramer, has been reported thus far. In this report, we use structural analysis and computational design to convert this homotetramer into the smallest known α/β-heterotetramer. Computational screening of many possible sequence/structure combinations led efficiently to the design of short, 21-residue peptides that fold cooperatively and autonomously into a specific complex in solution. A 1.95 Å crystal structure reveals how steric complementarity and charge patterning encode heterospecificity. The first- and second-generation heterotetrameric miniproteins described here will be useful as simple models for the analysis of protein-protein interaction specificity and as structural platforms for the further elaboration of folding and function.

Single-molecule atomic force microscopy and spectroscopy were applied to detect molecular interactions stabilizing the structure of halorhodopsin (HR), a light-driven chloride pump from Halobacterium salinarum. Because of the high structural and sequence similarities between HR and bacteriorhodopsin, we compared their unfolding pathways and polypeptide regions that established structurally stable segments against unfolding. Unfolding pathways and structural segments stabilizing the proteins both exhibited a remarkably high similarity. This suggests that different amino acid compositions can establish structurally indistinguishable energetic barriers. These stabilizing domains rather result from comprehensive interactions of all amino acids within a structural region than from specific interactions. However, one additional unfolding barrier located within a short segment of helix E was detected for HR. This barrier correlated with a Pi-bulk interaction, which locally disrupts helix E and divides a structural stabilizing segment.

Three-Dimensional Structure and Regulation of the DNA-Dependent Protein Kinase Catalytic Subunit (DNA-PKcs) by Angel Rivera-Calzada; Joseph P. Maman; Laura Spagnolo; Laurence H. Pearl; Oscar Llorca (243-255).
DNA-PKcs is a large PI3-kinase-related protein kinase (PIKK) that plays a central role in DNA double-strand break (DSB) repair via nonhomologous end joining. Using cryo-electron microscopy we have now generated a ∼13 Å three-dimensional map of DNA-PKcs, revealing the overall architecture and topology of the 4128 residue polypeptide chain and allowing location of domains. The highly conserved C-terminal PIKK catalytic domain forms a central structure from which FAT and FATC domains protrude. Conformational changes observed in these domains on DNA binding suggest that they transduce DNA-induced conformational changes to the catalytic core and regulate kinase activity. The N-terminal segments form long curved tubular-shaped domains based on helical repeats to create interacting surfaces required for macromolecular assembly. Comparison of DNA-PKcs with another PIKK DNA repair factor, ATM, defines a common architecture for this important protein family

Assessment of the Robustness of a Serendipitous Zinc Binding Fold: Mutagenesis and Protein Grafting by Belinda K. Sharpe; Chu Kong Liew; Ann H. Kwan; Jackie A. Wilce; Merlin Crossley; Jacqueline M. Matthews; Joel P. Mackay (257-266).
Zinc binding motifs have received much attention in the area of protein design. Here, we have tested the suitability of a recently discovered nonnative zinc binding structure as a protein design scaffold. A series of multiple alanine mutants was created to investigate the minimal requirements for folding, and solution structures of these mutants showed that the original fold was maintained, despite changes in ∼50% of the sequence. We next attempted to transplant binding faces from chosen bimolecular interactions onto one of these mutants, and many of the resulting “chimeras” were shown to adopt a native-like fold. These results both highlight the robust nature of small zinc binding domains and underscore the complexity of designing functional proteins, even using such small, highly ordered scaffolds as templates.

Structural Studies of the Human PBAF Chromatin-Remodeling Complex by Andres E Leschziner; Bryan Lemon; Robert Tjian; Eva Nogales (267-275).
ATP-dependent chromatin remodeling is one of the central processes responsible for imparting fluidity to chromatin and thus regulating DNA transactions. Although knowledge on this process is accumulating rapidly, the basic mechanism (or mechanisms) by which the remodeling complexes alter the structure of a nucleosome is not yet understood. Structural information on these macromolecular machines should aid in interpreting the biochemical and genetic data; to this end, we have determined the structure of the human PBAF ATP-dependent chromatin-remodeling complex preserved in negative stain by electron microscopy and have mapped the nucleosome binding site using two-dimensional (2D) image analysis. PBAF has an overall C-shaped architecture—with a larger density to which two smaller knobs are attached—surrounding a central cavity; one of these knobs appears to be flexible and occupies different positions in each of the structures determined. The 2D analysis of PBAF:nucleosome complexes indicates that the nucleosome binds in the central cavity.

Structure and Phosphatidylinositol-(3,4)- Bisphosphate Binding of the C-Terminal PH Domain of Human Pleckstrin by Christian Edlich; Gunter Stier; Bernd Simon; Michael Sattler; Claudia Muhle-Goll (277-286).
Pleckstrin is the major target of protein kinase C (PKC) in blood platelets. Its phosphorylation triggers responses that ultimately lead to platelet activation and blood clot formation. Pleckstrin consists of three domains: a pleckstrin homology (PH) domain at both termini and a central DEP (Dishevelled, Egl-1, Pleckstrin) domain. Here, we report the solution nuclear magnetic resonance (NMR) structure of the C-terminal PH domain (C-PH) of human pleckstrin-1. We show that this PH domain binds phosphatidylinositol-3,4-bisphosphate (PtdIns(3,4)P2) with high specificity in protein lipid overlay assays. Using NMR titration experiments and mutational analysis, residues involved in binding to PtdIns(3,4)P2 are identified. The binding site is formed by a patch of basic residues from the β1 and β2 strands and the β1−β2 loop. Since PtdIns(3,4)P2 is an important signaling molecule in platelets, our data suggest a C-PH dependent regulation of pleckstrin function in response to PtdIns(3,4)P2.

Small-Angle X-Ray Scattering Reveals the Solution Structure of the Full-Length DNA Gyrase A Subunit by Lionel Costenaro; J. Günter Grossmann; Christine Ebel; Anthony Maxwell (287-296).
DNA gyrase is the topoisomerase uniquely able to actively introduce negative supercoils into DNA. Vital in all bacteria, but absent in humans, this enzyme is a successful target for antibacterial drugs. From biophysical experiments in solution, we report the low-resolution structure of the full-length A subunit (GyrA). Analytical ultracentrifugation shows that GyrA is dimeric, but nonglobular. Ab initio modeling from small-angle X-ray scattering allows us to retrieve the molecular envelope of GyrA and thereby the organization of its domains. The available crystallographic structure of the amino-terminal domain (GyrA59) forms a dimeric core, and two additional pear-shaped densities closely flank it in an unexpected position. Each accommodates very well a carboxyl-terminal domain (GyrA-CTD) built from a homologous crystallographic structure. The uniqueness of gyrase is due to the ability of the GyrA-CTDs to wrap DNA. Their position within the GyrA structure strongly suggests a large conformation change of the enzyme upon DNA binding.

Magnitude of the Hydrophobic Effect at Central versus Peripheral Sites in Protein-Protein Interfaces by Yili Li; Yuping Huang; Chittoor P. Swaminathan; Sandra J. Smith-Gill; Roy A. Mariuzza (297-307).
Hydrophobic interactions are essential for stabilizing protein-protein complexes, whose interfaces generally consist of a central cluster of hot spot residues surrounded by less important peripheral residues. According to the O-ring hypothesis, a condition for high affinity binding is solvent exclusion from interacting residues. This hypothesis predicts that the hydrophobicity at the center is significantly greater than at the periphery, which we estimated at 21 cal mol−1 Å−2. To measure the hydrophobicity at the center, structures of an antigen-antibody complex where a buried phenylalanine was replaced by smaller hydrophobic residues were determined. By correlating structural changes with binding free energies, we estimate the hydrophobicity at this central site to be 46 cal mol−1 Å−2, twice that at the periphery. This context dependence of the hydrophobic effect explains the clustering of hot spots at interface centers and has implications for hot spot prediction and the design of small molecule inhibitors.

An Inflammatory Role for the Mammalian Carboxypeptidase Inhibitor Latexin: Relationship to Cystatins and the Tumor Suppressor TIG1 by Anna Aagaard; Pawel Listwan; Nathan Cowieson; Thomas Huber; Timothy Ravasi; Christine A. Wells; Jack U. Flanagan; Stuart Kellie; David A. Hume; Bostjan Kobe; Jennifer L. Martin (309-317).
Latexin, the only known mammalian carboxypeptidase inhibitor, has no detectable sequence similarity with plant and parasite inhibitors, but it is related to a human putative tumor suppressor protein, TIG1. Latexin is expressed in the developing brain, and we find that it plays a role in inflammation, as it is expressed at high levels and is inducible in macrophages in concert with other protease inhibitors and potential protease targets. The crystal structure of mouse latexin, solved at 1.83 Å resolution, shows no structural relationship with other carboxypeptidase inhibitors. Furthermore, despite a lack of detectable sequence duplication, the structure incorporates two topologically analogous domains related by pseudo two-fold symmetry. Surprisingly, these domains share a cystatin fold architecture found in proteins that inhibit cysteine proteases, suggesting an evolutionary and possibly functional relationship. The structure of the tumor suppressor protein TIG1 was modeled, revealing its putative membrane binding surface.

Probing the Supramodular Architecture of a Multidomain Protein: The Structure of Syntenin in Solution by Tomasz Cierpicki; John H. Bushweller; Zygmunt S. Derewenda (319-327).
Full understanding of the mechanism of function of multidomain proteins is dependent on our knowledge of their supramodular architecture in solution. This is a nontrivial task for both X-ray crystallography and NMR, because intrinsic flexibility makes crystallization of these proteins difficult, while their size creates a challenge for NMR. Here, we describe synergistic application of data derived from X-ray crystallography and NMR residual dipolar couplings (RDCs) to address the question of the supramodular structure of a two-domain protein, syntenin. Syntenin is a 32 kDa molecule containing two PDZ domains and is involved in cytoskeleton-membrane organization. We show that the mutual disposition of the PDZ domains clearly differs from that seen in the crystal structure, and we provide evidence that N- and C-terminal fragments of syntenin, hitherto presumed to lack ordered structure, contain folded structural elements in the full-length protein in contact with the PDZ tandem.

dUTPase as a Platform for Antimalarial Drug Design: Structural Basis for the Selectivity of a Class of Nucleoside Inhibitors by Jean L. Whittingham; Isabel Leal; Corinne Nguyen; Ganasan Kasinathan; Emma Bell; Andrew F. Jones; Colin Berry; Agustin Benito; Johan P. Turkenburg; Eleanor J. Dodson; Luis M. Ruiz Perez; Anthony J. Wilkinson; Nils Gunnar Johansson; Reto Brun; Ian H. Gilbert; Dolores Gonzalez Pacanowska; Keith S. Wilson (329-338).