Structure (v.14, #1)

Electron-Induced Enzyme Activation by Andrea T. Hadfield (1-2).
In this issue of Structure, report structures of a bacterial diheme cyctochrome c peroxidase in an oxidized inactive and an activated reduced state. The two structures give insight into the activation at one heme through reduction of the other.

The Double Life of Aconitase by Peter J. Artymiuk; Jeffrey Green (2-4).
In this issue of Structure, Volbeda, Fontecilla-Camps, and colleagues () present the first structure of a eukaryotic cytoplasmic aconitase (cAcn). Their work casts new light on the crucial RNA binding function of some of the members of the aconitase superfamily.

Topological Determinants of Protein Domain Swapping by Feng Ding; Kirk C. Prutzman; Sharon L. Campbell; Nikolay V. Dokholyan (5-14).
Protein domain swapping has been repeatedly observed in a variety of proteins and is believed to result from destabilization due to mutations or changes in environment. Based on results from our studies and others, we propose that structures of the domain-swapped proteins are mainly determined by their native topologies. We performed molecular dynamics simulations of seven different proteins, known to undergo domain swapping experimentally, under mildly denaturing conditions and found in all cases that the domain-swapped structures can be recapitulated by using protein topology in a simple protein model. Our studies further indicated that, in many cases, domain swapping occurs at positions around which the protein tends to unfold prior to complete unfolding. This, in turn, enabled prediction of protein structural elements that are responsible for domain swapping. In particular, two distinct domain-swapped dimer conformations of the focal adhesion targeting domain of focal adhesion kinase were predicted computationally and were supported experimentally by data obtained from NMR analyses.

The Mechanism of HIV-1 Core Assembly: Insights from Three-Dimensional Reconstructions of Authentic Virions by John A.G. Briggs; Kay Grünewald; Bärbel Glass; Friedrich Förster; Hans-Georg Kräusslich; Stephen D. Fuller (15-20).
Infectious HIV particles contain a characteristic cone-shaped core encasing the viral RNA and replication proteins. The core exhibits significant heterogeneity in size and shape, yet consistently forms a well-defined structure. The mechanism by which the core is assembled in the maturing virion remains poorly understood. Using cryo-electron tomography, we have produced three-dimensional reconstructions of authentic, unstained HIV-1. These reveal the viral morphology with unprecedented clarity and suggest the following mechanism for core formation inside the extracellular virion: core growth initiates at the narrow end of the cone and proceeds toward the distal side of the virion until limited by the viral membrane. Curvature and closure of the broad end of the core are then directed by the inner surface of the viral membrane. This mechanism accommodates significant flexibility in lattice growth while ensuring the closure of cores of variable size and shape.

The Structure of the N-Terminal Domain of the Fragile X Mental Retardation Protein: A Platform for Protein-Protein Interaction by Andres Ramos; David Hollingworth; Salvatore Adinolfi; Marie Castets; Geoff Kelly; Thomas A. Frenkiel; Barbara Bardoni; Annalisa Pastore (21-31).
FMRP, whose lack of expression causes the X-linked fragile X syndrome, is a modular RNA binding protein thought to be involved in posttranslational regulation. We have solved the structure in solution of the N-terminal domain of FMRP (NDF), a functionally important region involved in multiple interactions. The structure consists of a composite fold comprising two repeats of a Tudor motif followed by a short α helix. The interactions between the three structural elements are essential for the stability of the NDF fold. Although structurally similar, the two repeats have different dynamic and functional properties. The second, more flexible repeat is responsible for interacting both with methylated lysine and with 82-FIP, one of the FMRP nuclear partners. NDF contains a 3D nucleolar localization signal, since destabilization of its fold leads to altered nucleolar localization of FMRP. We suggest that the NDF composite fold determines an allosteric mechanism that regulates the FMRP functions.

The Structures of the Thrombospondin-1 N-Terminal Domain and Its Complex with a Synthetic Pentameric Heparin by Kemin Tan; Mark Duquette; Jin-huan Liu; Rongguang Zhang; Andrzej Joachimiak; Jia-huai Wang; Jack Lawler (33-42).
The N-terminal domain of thrombospondin-1 (TSPN-1) mediates the protein's interaction with (1) glycosaminoglycans, calreticulin, and integrins during cellular adhesion, (2) low-density lipoprotein receptor-related protein during uptake and clearance, and (3) fibrinogen during platelet aggregation. The crystal structure of TSPN-1 to 1.8 Å resolution is a β sandwich with 13 antiparallel β strands and 1 irregular strand-like segment. Unique structural features of the N- and C-terminal regions, and the disulfide bond location, distinguish TSPN-1 from the laminin G domain and other concanavalin A-like lectins/glucanases superfamily members. The crystal structure of the complex of TSPN-1 with heparin indicates that residues R29, R42, and R77 in an extensive positively charged patch at the bottom of the domain specifically associate with the sulfate groups of heparin. The TSPN-1 structure and identified adjacent linker region provide a structural framework for the analysis of the TSPN domain of various molecules, including TSPs, NELLs, many collagens, TSPEAR, and kielin.

The coiled coil is a widespread motif involved in oligomerization and protein-protein interactions, but the structural requirements for binding to target proteins are poorly understood. To address this question, we measured binding of tropomyosin, the prototype coiled coil, to actin as a model system. Tropomyosin binds to the actin filament and cooperatively regulates its function. Our results support the hypothesis that coiled-coil domains that bind to other proteins are flexible. We made mutations that alter interface packing and stability as well as mutations in surface residues in a postulated actin binding site. Actin affinity, measured by cosedimentation, was correlated with coiled-coil stability and local instability and side chain flexibility, analyzed with circular dichroism and fluorescence spectroscopy. The flexibility from interruptions in the stable coiled-coil interface is essential for actin binding. The surface residues in a postulated actin binding site participate in actin binding when the coiled coil within it is poorly packed.

A Second FMN Binding Site in Yeast NADPH-Cytochrome P450 Reductase Suggests a Mechanism of Electron Transfer by Diflavin Reductases by David C. Lamb; Youngchang Kim; Liudmila V. Yermalitskaya; Valery N. Yermalitsky; Galina I. Lepesheva; Steven L. Kelly; Michael R. Waterman; Larissa M. Podust (51-61).
NADPH-cytochrome P450 reductase transfers two reducing equivalents derived from a hydride ion of NADPH via FAD and FMN to the large family of microsomal cytochrome P450 monooxygenases in one-electron transfer steps. The mechanism of electron transfer by diflavin reductases remains elusive and controversial. Here, we determined the crystal structure of truncated yeast NADPH-cytochrome P450 reductase, which is functionally active toward its physiological substrate cytochrome P450, and discovered a second FMN binding site at the interface of the connecting and FMN binding domains. The two FMN binding sites have different accessibilities to the bulk solvent and different amino acid environments, suggesting stabilization of different electronic structures of the reduced flavin. Since only one FMN cofactor is required for function, a hypothetical mechanism of electron transfer is discussed that proposes shuttling of a single FMN between these two sites coupled with the transition between two semiquinone forms, neutral (blue) and anionic (red).

Mapping the Structure and Function of the E1 and E2 Glycoproteins in Alphaviruses by Suchetana Mukhopadhyay; Wei Zhang; Stefan Gabler; Paul R. Chipman; Ellen G. Strauss; James H. Strauss; Timothy S. Baker; Richard J. Kuhn; Michael G. Rossmann (63-73).
The 9 Å resolution cryo-electron microscopy map of Sindbis virus presented here provides structural information on the polypeptide topology of the E2 protein, on the interactions between the E1 and E2 glycoproteins in the formation of a heterodimer, on the difference in conformation of the two types of trimeric spikes, on the interaction between the transmembrane helices of the E1 and E2 proteins, and on the conformational changes that occur when fusing with a host cell. The positions of various markers on the E2 protein established the approximate topology of the E2 structure. The largest conformational differences between the icosahedral surface spikes at icosahedral 3-fold and quasi-3-fold positions are associated with the monomers closest to the 5-fold axes. The long E2 monomers, containing the cell receptor recognition motif at their extremities, are shown to rotate by about 180° and to move away from the center of the spikes during fusion.

Structure and Interactions at the Viral Surface of the Envelope Protein E1 of Semliki Forest Virus by Alain Roussel; Julien Lescar; Marie-Christine Vaney; Gisela Wengler; Gerd Wengler; Félix A. Rey (75-86).
Semliki Forest virus (SFV) is enveloped by a lipid bilayer enclosed within a glycoprotein cage made by glycoproteins E1 and E2. E1 is responsible for inducing membrane fusion, triggered by exposure to the acidic environment of the endosomes. Acidic pH induces E1/E2 dissociation, allowing E1 to interact with the target membrane, and, at the same time, to rearrange into E1 homotrimers that drive the membrane fusion reaction. We previously reported a preliminary Cα trace of the monomeric E1 glycoprotein ectodomain and its organization on the virus particle. We also reported the 3.3 Å structure of the trimeric, fusogenic conformation of E1. Here, we report the crystal structure of monomeric E1 refined to 3 Å resolution and describe the amino acids involved in contacts in the virion. These results identify the major determinants for the E1/E2 icosahedral shell formation and open the way to rational mutagenesis approaches to shed light on SFV assembly.

Mammalian coronin-1 is preferentially expressed in hematopoietic cells and plays a poorly understood role in the dynamic reorganization of the actin cytoskeleton. Sequence analysis of coronin-1 revealed five WD40 repeats that were predicted to form a β propeller. They are followed by a 130 residue extension and a 30 residue leucine zipper domain that is responsible for multimerization of the protein. Here, we present the crystal structure of murine coronin-1 without the leucine zipper at 1.75 Å resolution. Coronin-1 forms a seven-bladed β propeller composed of the five predicted WD40 repeats and two additional blades that lack any homology to the canonical WD40 motif. The C-terminal extension adopts an extended conformation, packs tightly against the bottom surface of the propeller, and is likely to be required for the structural stability of the propeller. Analysis of charged and conserved surface residues delineate possible binding sites for F-actin on the β propeller.

Protection factors obtained from equilibrium hydrogen exchange experiments are an important source of structural information on both native and nonnative states of proteins. We present a method for determining ensembles of protein structures by using hydrogen exchange data as restraints in molecular dynamics simulations in conjunction with an empirical force-field. The method is applied to determine the ensemble of structures representing the native state of chymotrypsin inhibitor 2 (CI2), including the rare, large fluctuations responsible for hydrogen exchange.

Activation and Catalysis of the Di-Heme Cytochrome c Peroxidase from Paracoccus pantotrophus by Aude Echalier; Celia F. Goodhew; Graham W. Pettigrew; Vilmos Fülöp (107-117).
Bacterial cytochrome c peroxidases contain an electron transferring (E) heme domain and a peroxidatic (P) heme domain. All but one of these enzymes are isolated in an inactive oxidized state and require reduction of the E heme by a small redox donor protein in order to activate the P heme. Here we present the structures of the inactive oxidized and active mixed valence enzyme from Paracoccus pantotrophus. Chain flexibility in the former, as expressed by the crystallographic temperature factors, is strikingly distributed in certain loop regions, and these coincide with the regions of conformational change that occur in forming the active mixed valence enzyme. On the basis of these changes, we postulate a series of events that occur to link the trigger of the electron entering the E heme from either pseudoazurin or cytochrome c550 and the dissociation of a coordinating histidine at the P heme, which allows substrate access.

Structural Switch of the γ Subunit in an Archaeal aIF2αγ Heterodimer by Laure Yatime; Yves Mechulam; Sylvain Blanquet; Emmanuelle Schmitt (119-128).
Eukaryotic and archaeal initiation factors 2 (e/aIF2) are heterotrimeric proteins (αβγ) supplying the small subunit of the ribosome with methionylated initiator tRNA. This study reports the crystallographic structure of an aIF2αγ heterodimer from Sulfolobus solfataricus bound to Gpp(NH)p-Mg2+. aIF2γ is in a closed conformation with the G domain packed on domains II and III. The C-terminal domain of aIF2α interacts with domain II of aIF2γ. Conformations of the two switch regions involved in GTP binding are similar to those encountered in an EF1A:GTP:Phe-tRNAPhe complex. Comparison with the EF1A structure suggests that only the γ subunit of the aIF2αγ heterodimer contacts tRNA. Because the α subunit markedly reinforces the affinity of tRNA for the γ subunit, a contribution of the α subunit to the switch movements observed in the γ structure is considered.

Crystal Structure of Human Iron Regulatory Protein 1 as Cytosolic Aconitase by Jérôme Dupuy; Anne Volbeda; Philippe Carpentier; Claudine Darnault; Jean-Marc Moulis; Juan Carlos Fontecilla-Camps (129-139).
Iron regulatory proteins (IRPs) control the translation of proteins involved in iron uptake, storage and utilization by binding to specific noncoding sequences of the corresponding mRNAs known as iron-responsive elements (IREs). This strong interaction assures proper iron homeostasis in animal cells under iron shortage. Conversely, under iron-replete conditions, IRP1 binds a [4Fe-4S] cluster and functions as cytosolic aconitase. Regulation of the balance between the two IRP1 activities is complex, and it does not depend only on iron availability. Here, we report the crystal structure of human IRP1 in its aconitase form. Comparison with known structures of homologous enzymes reveals well-conserved folds and active site environments with significantly different surface shapes and charge distributions. The specific features of human IRP1 allow us to propose a tentative model of an IRP1-IRE complex that agrees with a range of previously obtained data.

Dynamic Binding of PKA Regulatory Subunit RIα by Justin Gullingsrud; Choel Kim; Susan S. Taylor; J. Andrew McCammon (141-149).
Recent crystal structures have revealed that regulatory subunit RIα of PKA undergoes a dramatic conformational change upon complex formation with the catalytic subunit. Molecular dynamics studies were initiated to elucidate the contributions of intrinsic conformational flexibility and interactions with the catalytic subunit in formation and stabilization of the complex. Simulations of a single RIα nucleotide binding domain (NBD), missing cAMP, showed that its C helix spontaneously occupies two distinct conformations: either packed against the nucleotide binding domain as in its cAMP bound structure, or extended into an intermediate form resembling that of the holoenzyme structure. C helix extension was not seen in a simulation of either RIα NBD. In a model complex containing both NBDs and the catalytic subunit, well-conserved residues at the interface between the NBDs in the cAMP bound form were found to stabilize the complex through contacts with the catalytic subunit. The model structure is consistent with available experimental data.

Multipurpose MRG Domain Involved in Cell Senescence and Proliferation Exhibits Structural Homology to a DNA-Interacting Domain by Brian R. Bowman; Carmen M. Moure; Bhakti M. Kirtane; Robert L. Welschhans; Kaoru Tominaga; Olivia M. Pereira-Smith; Florante A. Quiocho (151-158).
The ubiquitous MRG/MORF family of proteins is involved in cell senescence, or the terminal loss of proliferative potential, a model for aging and tumor suppression at the cellular level. These proteins are defined by the ∼20 kDa MRG domain that binds a plethora of transcriptional regulators and chromatin-remodeling factors, including the histone deacetylase transcriptional corepressor mSin3A and the novel nuclear protein PAM14, and they are also known components of the Tip60/NuA4 complex via interactions with the MRG binding protein (MRGBP). We present here the crystal structure of a prototypic MRG domain from human MRG15 whose core consists of two orthogonal helix hairpins. Despite the lack of sequence similarity, the core structure has surprisingly striking homology to a DNA-interacting domain of the tyrosine site-specific recombinases XerD, λ integrase, and Cre. Site-directed mutagenesis studies based on the X-ray structure and bioinformatics identified key residues involved in the binding of PAM14 and MRGBP.

Structure of the Forkhead Domain of FOXP2 Bound to DNA by James C. Stroud; Yongqing Wu; Darren L. Bates; Aidong Han; Katja Nowick; Svante Paabo; Harry Tong; Lin Chen (159-166).
FOXP (FOXP1–4) is a newly defined subfamily of the forkhead box (FOX) transcription factors. A mutation in the FOXP2 forkhead domain cosegregates with a severe speech disorder, whereas several mutations in the FOXP3 forkhead domain are linked to the IPEX syndrome in human and a similar autoimmune phenotype in mice. Here we report a 1.9 Å crystal structure of the forkhead domain of human FOXP2 bound to DNA. This structure allows us to revise the previously proposed DNA recognition mechanism and provide a unifying model of DNA binding for the FOX family of proteins. Our studies also reveal that the FOXP2 forkhead domain can form a domain-swapped dimer, made possible by a strategic substitution of a highly conserved proline in conventional FOX proteins with alanine in the P subfamily. Disease-causing mutations in FOXP2 and FOXP3 map either to the DNA binding surface or the domain-swapping dimer interface, functionally corroborating the crystal structure.