Structure (v.15, #1)

Searchlight on Domains by Monica Riley (1-2).
In this issue of Structure, examine in detail the functions of selected domains within proteins both when they are alone and when in combination with others. Domain function is relevant to molecular evolution and to annotation of proteins known only by sequence.

Tying the Knot That Binds by Lisa M. Gloss (2-4).
The recent determination of protein structures with knots in their backbone topology has defied previous conventional wisdom. How proteins can fold with a knot is an intriguing question that has been explored for YibK from Haemophilus influenzae in this issue of Structure ().

Bypassing Translation Initiation by Christopher U.T. Hellen (4-6).
A high-resolution cryo-EM reconstruction of a ribosome-bound dicistrovirus IRES () and the crystal structure of its ribosome binding domain () provide new insights into an exceptional eukaryotic translation mechanism.

An increasing number of structural studies of large macromolecular complexes, both in X-ray crystallography and cryo-electron microscopy, have resulted in intermediate-resolution (5–10 Å) density maps. Despite being limited in resolution, significant structural and functional information may be extractable from these maps. To aid in the analysis and annotation of these complexes, we have developed SSEhunter, a tool for the quantitative detection of α helices and β sheets. Based on density skeletonization, local geometry calculations, and a template-based search, SSEhunter has been tested and validated on a variety of simulated and authentic subnanometer-resolution density maps. The result is a robust, user-friendly approach that allows users to quickly visualize, assess, and annotate intermediate-resolution density maps. Beyond secondary structure element identification, the skeletonization algorithm in SSEhunter provides secondary structure topology, which is potentially useful in leading to structural models of individual molecular components directly from the density.

Structural and Thermodynamic Principles of Viral Packaging by Anton S. Petrov; Stephen C. Harvey (21-27).
Packaging of genetic material inside a capsid is one of the major processes in the lifecycle of bacteriophages. To establish the basic principles of packing double-stranded DNA into a phage, we present a low-resolution model of bacteriophage ϕ29 and report simulations of DNA packaging. The simulations show excellent agreement with available experimental data, including the forces of packaging and the average structures seen in cryo-electron microscopy. The conformation of DNA inside the bacteriophage is primarily determined by the shape of the capsid and the elastic properties of DNA, but the energetics of packaging are dominated by electrostatic repulsions and the large entropic penalty associated with DNA confinement. In this slightly elongated capsid, the DNA assumes a folded toroidal conformation, rather than a coaxial spool. The model can be used to study packaging of other bacteriophages with different shapes under a range of environmental conditions.
Keywords: MICROBIOL;

Atomic Resolution Structures of Rieske Iron-Sulfur Protein: Role of Hydrogen Bonds in Tuning the Redox Potential of Iron-Sulfur Clusters by Derrick J. Kolling; Joseph S. Brunzelle; SangMoon Lhee; Antony R. Crofts; Satish K. Nair (29-38).
The Rieske [2Fe-2S] iron-sulfur protein of cytochrome bc 1 functions as the initial electron acceptor in the rate-limiting step of the catalytic reaction. Prior studies have established roles for a number of conserved residues that hydrogen bond to ligands of the [2Fe-2S] cluster. We have constructed site-specific variants at two of these residues, measured their thermodynamic and functional properties, and determined atomic resolution X-ray crystal structures for the native protein at 1.2 Å resolution and for five variants (Ser-154→Ala, Ser-154→Thr, Ser-154→Cys, Tyr-156→Phe, and Tyr-156→Trp) to resolutions between 1.5 Å and 1.1 Å. These structures and complementary biophysical data provide a molecular framework for understanding the role hydrogen bonds to the cluster play in tuning thermodynamic properties, and hence the rate of this bioenergetic reaction. These studies provide a detailed structure-function dissection of the role of hydrogen bonds in tuning the redox potentials of [2Fe-2S] clusters.

Actin structure is of intense interest in biology due to its importance in cell function and motility mediated by the spatial and temporal regulation of actin monomer-filament interconversions in a wide range of developmental and disease states. Despite this interest, the structure of many functionally important actin forms has eluded high-resolution analysis. Due to the propensity of actin monomers to assemble into filaments structural analysis of Mg-bound actin monomers has proven difficult, whereas high-resolution structures of actin with a diverse array of ligands that preclude polymerization have been quite successful. In this work, we provide a high-resolution structural model of the Mg-ATP-actin monomer using a combination of computational methods and experimental footprinting data that we have previously published. The key conclusion of this study is that the structure of the nucleotide binding cleft defined by subdomains 2 and 4 is essentially closed, with specific contacts between two subdomains predicted by the data.
Keywords: CELLBIO;

All-Atom Ab Initio Folding of a Diverse Set of Proteins by Jae Shick Yang; William W. Chen; Jeffrey Skolnick; Eugene I. Shakhnovich (53-63).
Natural proteins fold to a unique, thermodynamically dominant state. Modeling of the folding process and prediction of the native fold of proteins are two major unsolved problems in biophysics. Here, we show successful all-atom ab initio folding of a representative diverse set of proteins by using a minimalist transferable-energy model that consists of two-body atom-atom interactions, hydrogen bonding, and a local sequence-energy term that models sequence-specific chain stiffness. Starting from a random coil, the native-like structure was observed during replica exchange Monte Carlo (REMC) simulation for most proteins regardless of their structural classes; the lowest energy structure was close to native—in the range of 2–6 Å root-mean-square deviation (rmsd). Our results demonstrate that the successful folding of a protein chain to its native state is governed by only a few crucial energetic terms.

Structure of the Bateman2 Domain of Yeast Snf4: Dimeric Association and Relevance for AMP Binding by Michael J. Rudolph; Gabriele A. Amodeo; Surtaj H. Iram; Seung-Pyo Hong; Giorgia Pirino; Marian Carlson; Liang Tong (65-74).
AMP-activated protein kinase (AMPK) is a central regulator of energy homeostasis in mammals. AMP is believed to control the activity of AMPK by binding to the γ subunit of this heterotrimeric enzyme. This subunit contains two Bateman domains, each of which is composed of a tandem pair of cystathionine β-synthase (CBS) motifs. No structural information is currently available on this subunit, and the molecular basis for its interactions with AMP is not well understood. We report here the crystal structure at 1.9 Å resolution of the Bateman2 domain of Snf4, the γ subunit of the yeast ortholog of AMPK. The structure revealed a dimer of the Bateman2 domain, and this dimerization is supported by our light-scattering, mutagenesis, and biochemical studies. There is a prominent pocket at the center of this dimer, and most of the disease-causing mutations are located in or near this pocket.
Keywords: SIGNALING;

DNA Recognition Mechanism of the ONECUT Homeodomain of Transcription Factor HNF-6 by Daisuke Iyaguchi; Min Yao; Nobuhisa Watanabe; Jun Nishihira; Isao Tanaka (75-83).
Hepatocyte nuclear factor-6 (HNF-6), a liver-enriched transcription factor, controls the development of various tissues, such as the pancreas and liver, and regulates the expression of several hepatic genes. This protein belongs to the ONECUT class of homeodomain proteins and contains a bipartite DNA-binding domain composed of a single cut domain and a characteristic homeodomain. This transcription factor has two distinct modes of DNA binding and transcriptional activation that use different coactivators depending on the target gene. The crystal structure of the bipartite DNA-binding domain of HNF-6α complexed with the HNF-6-binding site of the TTR promoter revealed the DNA recognition mechanism of this protein. Comparing our structure with the DNA-free structure of HNF-6 or the structure of Oct-1, we discuss characteristic features associated with DNA binding and the structural basis for the dual mode of action of this protein, and we suggest a strategy for variability of transcriptional activation of the target gene.

During evolution, many new proteins have been formed by the process of gene duplication and combination. The genes involved in this process usually code for whole domains. Small proteins contain one domain; medium and large proteins contain two or more domains. We have compared homologous domains that occur in both one-domain proteins and multidomain proteins. We have determined (1) how the functions of the individual domains in the multidomain proteins combine to produce their overall functions and (2) the extent to which these functions are similar to those in the one-domain homologs. We describe how domain combinations increase the specificity of enzymes; act as links between domains that have functional roles; regulate activity; combine within one chain functions that can act either independently, in concert or in new contexts; and provide the structural framework for the evolution of entirely new functions.
Keywords: EVO_ECOL;

Divergent Substrate-Binding Mechanisms Reveal an Evolutionary Specialization of Eukaryotic Prefoldin Compared to Its Archaeal Counterpart by Jaime Martín-Benito; Juan Gómez-Reino; Peter C. Stirling; Victor F. Lundin; Paulino Gómez-Puertas; Jasminka Boskovic; Pablo Chacón; José J. Fernández; José Berenguer; Michel R. Leroux; José M. Valpuesta (101-110).
Prefoldin (PFD) is a molecular chaperone that stabilizes and then delivers unfolded proteins to a chaperonin for facilitated folding. The PFD hexamer has undergone an evolutionary change in subunit composition, from two PFDα and four PFDβ subunits in archaea to six different subunits (two α-like and four β-like subunits) in eukaryotes. Here, we show by electron microscopy that PFD from the archaeum Pyrococcus horikoshii (PhPFD) selectively uses an increasing number of subunits to interact with nonnative protein substrates of larger sizes. PhPFD stabilizes unfolded proteins by interacting with the distal regions of the chaperone tentacles, a mechanism different from that of eukaryotic PFD, which encapsulates its substrate inside the cavity. This suggests that although the fundamental functions of archaeal and eukaryal PFD are conserved, their mechanism of substrate interaction have diverged, potentially reflecting a narrower range of substrates stabilized by the eukaryotic PFD.
Keywords: PROTEINS;

α/β-Knotted proteins are an extraordinary example of biological self-assembly; they contain a deep topological trefoil knot formed by the backbone polypeptide chain. Evidence suggests that all are dimeric and function as methyltransferases, and the deep knot forms part of the active site. We investigated the significance of the dimeric structure of the α/β-knot protein, YibK, from Haemophilus influenzae by the design and engineering of monomeric versions of the protein, followed by examination of their structural, functional, stability, and kinetic folding properties. Monomeric forms of YibK display similar characteristics to an intermediate species populated during the formation of the wild-type dimer. However, a notable loss in structure involving disruption to the active site, rendering it incapable of cofactor binding, is observed in monomeric YibK. Thus, dimerization is vital for preservation of the native structure and, therefore, activity of the protein.

SR Protein Kinase 1 Is Resilient to Inactivation by Jacky Chi Ki Ngo; Justin Gullingsrud; Kayla Giang; Melinda Jean Yeh; Xiang-Dong Fu; Joseph A. Adams; J. Andrew McCammon; Gourisankar Ghosh (123-133).
SR protein kinase 1 (SRPK1) is a constitutively active kinase, which processively phosphorylates multiple serines within its substrates, ASF/SF2. We describe crystallographic, molecular dynamics, and biochemical results that shed light on how SRPK1 preserves its constitutive active conformation. Our structure reveals that unlike other known active kinase structures, the activation loop remains in an active state without any specific intraprotein interactions. Moreover, SRPK1 remains active despite extensive mutation to the activation segment. Molecular dynamics simulations reveal that SRPK1 partially absorbs the effect of mutations by forming compensatory interactions that maintain a catalytically competent chemical environment. Furthermore, SRPK1 is similarly resistant to deletion of its spacer loop region. Based upon a model of SRPK1 bound to a segment encompassing the docking motif and active-site peptide of ASF/SF2, we suggest a mechanism for processive phosphorylation and propose that the atypical resiliency we observed is critical for SRPK1's processive activity.