Structure (v.17, #12)

In This Issue (ix-x).

Expanding the Ribosomal Universe by Jonathan D. Dinman; Terri Goss Kinzy (1547-1548).
In this issue of Structure, Taylor et al. (2009) present the most complete model of an eukaryotic ribosome to date. This achievement represents a critical milestone along the path to structurally defining the unique aspects of the eukaryotic protein synthetic machinery.

Insights into Resistance against Lincosamide Antibiotics by Jesse A. Sundlov; Andrew M. Gulick (1549-1550).
Bacteria utilize multiple strategies to circumvent antibiotics, producing broad specificity exporters or enzymes that catalyze the modification of either antibiotics or their targets. A report in this issue of Structure provides the structural and catalytic mechanisms of LinB, an adenylyltransferase of E. faecium that confers resistance to the lincosamide antibiotic clindamycin.

Cyclic Nucleotide Binding GAF Domains from Phosphodiesterases: Structural and Mechanistic Insights by Clemens C. Heikaus; Jayvardhan Pandit; Rachel E. Klevit (1551-1557).
GAF domains regulate the catalytic activity of certain vertebrate cyclic nucleotide phosphodiesterases (PDEs) by allosteric, noncatalytic binding of cyclic nucleotides. GAF domains arranged in tandem are found in PDE2, −5, −6, −10, and −11, all of which regulate the cellular concentrations of the second messengers cAMP and/or cGMP. Nucleotide binding to GAF domains affects the overall conformation and the catalytic activity of full-length PDEs. The cyclic nucleotide-bound GAF domains from PDE2, −5, −6, and −10 all adopt a conserved fold but show subtle differences within the binding pocket architecture that account for a large range of nucleotide affinities and selectivity. NMR data and details from the structure of full-length nucleotide-free PDE2A reveal the dynamic nature and magnitude of the conformational change that accompanies nucleotide binding. The discussed GAF domain structures further reveal differences in dimerization properties and highlight the structural diversity within GAF domain-containing PDEs.

E Pluribus Tres: The 2009 Nobel Prize in Chemistry by Charles W. Carter (1558-1561).
This year's Nobel Prize in Chemistry celebrates a multitude of research areas, making the difficult selection of those most responsible for providing atomic details of the nanomachine that makes proteins according to genetic instructions. The Ribosome and RNA polymerase (recognized in 2006) structures highlight a puzzling asymmetry at the origins of biology.

Averaging of Electron Subtomograms and Random Conical Tilt Reconstructions through Likelihood Optimization by Sjors H.W. Scheres; Roberto Melero; Mikel Valle; Jose-Maria Carazo (1563-1572).
The reference-free averaging of three-dimensional electron microscopy (3D-EM) reconstructions with empty regions in Fourier space represents a pressing problem in electron tomography and single-particle analysis. We present a maximum likelihood algorithm for the simultaneous alignment and classification of subtomograms or random conical tilt (RCT) reconstructions, where the Fourier components in the missing data regions are treated as hidden variables. The behavior of this algorithm was explored using tests on simulated data, while application to experimental data was shown to yield unsupervised class averages for subtomograms of groEL/groES complexes and RCT reconstructions of p53. The latter application served to obtain a reliable de novo structure for p53 that may resolve uncertainties about its quaternary structure.
Keywords: PROTEINS;

Crystallographic Insight into Collagen Recognition by Discoidin Domain Receptor 2 by Federico Carafoli; Dominique Bihan; Stavros Stathopoulos; Antonios D. Konitsiotis; Marc Kvansakul; Richard W. Farndale; Birgit Leitinger; Erhard Hohenester (1573-1581).
The discoidin domain receptors, DDR1 and DDR2, are widely expressed receptor tyrosine kinases that are activated by triple-helical collagen. They control important aspects of cell behavior and are dysregulated in several human diseases. The major DDR2-binding site in collagens I–III is a GVMGFO motif (O is hydroxyproline) that also binds the matricellular protein SPARC. We have determined the crystal structure of the discoidin domain of human DDR2 bound to a triple-helical collagen peptide. The GVMGFO motifs of two collagen chains are recognized by an amphiphilic pocket delimited by a functionally critical tryptophan residue and a buried salt bridge. Collagen binding results in structural changes of DDR2 surface loops that may be linked to the process of receptor activation. A comparison of the GVMGFO-binding sites of DDR2 and SPARC reveals a striking case of convergent evolution in collagen recognition.

Non-Native Interactions Are Critical for Mechanical Strength in PKD Domains by Julia R. Forman; Zu Thur Yew; Seema Qamar; Richard N. Sandford; Emanuele Paci; Jane Clarke (1582-1590).
Experimental observation has led to the commonly held view that native state protein topology is the principle determinant of mechanical strength. However, the PKD domains of polycystin-1 challenge this assumption: they are stronger than predicted from their native structure. Molecular dynamics simulations suggest that force induces rearrangement to an intermediate structure, with nonnative hydrogen bonds, that resists unfolding. Here we test this hypothesis directly by introducing mutations designed to prevent formation of these nonnative interactions. We find that these mutations, which only moderately destabilize the native state, reduce the mechanical stability dramatically. The results demonstrate that nonnative interactions impart significant mechanical stability, necessary for the mechanosensor function of polycystin-1. Remarkably, such nonnative interactions result from force-induced conformational change: the PKD domain is strengthened by the application of force.

Comprehensive Molecular Structure of the Eukaryotic Ribosome by Derek J. Taylor; Batsal Devkota; Andrew D. Huang; Maya Topf; Eswar Narayanan; Andrej Sali; Stephen C. Harvey; Joachim Frank (1591-1604).
Despite the emergence of a large number of X-ray crystallographic models of the bacterial 70S ribosome over the past decade, an accurate atomic model of the eukaryotic 80S ribosome is still not available. Eukaryotic ribosomes possess more ribosomal proteins and ribosomal RNA than do bacterial ribosomes, which are implicated in extraribosomal functions in the eukaryotic cells. By combining cryo-EM with RNA and protein homology modeling, we obtained an atomic model of the yeast 80S ribosome complete with all ribosomal RNA expansion segments and all ribosomal proteins for which a structural homolog can be identified. Mutation or deletion of 80S ribosomal proteins can abrogate maturation of the ribosome, leading to several human diseases. We have localized one such protein unique to eukaryotes, rpS19e, whose mutations are associated with Diamond-Blackfan anemia in humans. Additionally, we characterize crucial interactions between the dynamic stalk base of the ribosome with eukaryotic elongation factor 2.

Many three-dimensional density maps of 70S ribosome at various functional states are available now in the Electron Microscopy DataBank at EBI. We used our new flexible-fitting approach to systematically analyze these maps to reveal the global conformational differences between the EM structures. Large-scale ratchet-like deformations were observed in an EM structure of the initiation complex and in some EM structures bound with EFG, RF3, and RRF. In most of them, the L1 stalk, which interacts with the tRNA molecule at the E site of ribosome and is considered to be involved in the release of the tRNA, was in “the blocking state” for the E-tRNA. Furthermore, we found that the EM structures bound with EFG or RRF were aligned in the conformational space, suggesting that the large-scale conformational changes of the 70S ribosome bound with these factors occur along a specific pathway in a concerted manner.

Unraveling the Allosteric Mechanism of Serine Protease Inhibition by an Antibody by Rajkumar Ganesan; Charles Eigenbrot; Yan Wu; Wei-Ching Liang; Steven Shia; Michael T. Lipari; Daniel Kirchhofer (1614-1624).
Recent structural studies have outlined the mechanism of protease inhibition by active site-directed antibodies. However, the molecular basis of allosteric inhibition by antibodies has been elusive. Here we report the 2.35 Å resolution structure of the trypsin-like serine protease hepatocyte growth factor activator (HGFA) in complex with the allosteric antibody Ab40, a potent inhibitor of HGFA catalytic activity. The antibody binds at the periphery of the substrate binding cleft and imposes a conformational change on the entire 99-loop (chymotrypsinogen numbering). The altered conformation of the 99-loop is incompatible with substrate binding due to the partial collapse of subsite S2 and the reorganization of subsite S4. Remarkably, a single residue deletion of Ab40 abolished inhibition of HGFA activity, commensurate with the reversal of the 99-loop conformation to its “competent” state. The results define an “allosteric switch” mechanism as the basis of protease inhibition by an allosteric antibody.
Keywords: PROTEINS;

Structure of HIV-1 Reverse Transcriptase with the Inhibitor β-Thujaplicinol Bound at the RNase H Active Site by Daniel M. Himmel; Karen A. Maegley; Tom A. Pauly; Joseph D. Bauman; Kalyan Das; Chhaya Dharia; Arthur D. Clark; Kevin Ryan; Michael J. Hickey; Robert A. Love; Stephen H. Hughes; Simon Bergqvist; Eddy Arnold (1625-1635).
Novel inhibitors are needed to counteract the rapid emergence of drug-resistant HIV variants. HIV-1 reverse transcriptase (RT) has both DNA polymerase and RNase H (RNH) enzymatic activities, but approved drugs that inhibit RT target the polymerase. Inhibitors that act against new targets, such as RNH, should be effective against all of the current drug-resistant variants. Here, we present 2.80 Å and 2.04 Å resolution crystal structures of an RNH inhibitor, β-thujaplicinol, bound at the RNH active site of both HIV-1 RT and an isolated RNH domain. β-thujaplicinol chelates two divalent metal ions at the RNH active site. We provide biochemical evidence that β-thujaplicinol is a slow-binding RNH inhibitor with noncompetitive kinetics and suggest that it forms a tropylium ion that interacts favorably with RT and the RNA:DNA substrate.

Drug-resistant mutations (DRMs) in HIV-1 protease are a major challenge to antiretroviral therapy. Protease-substrate interactions that are determined to be critical for native selectivity could serve as robust targets for drug design that are immune to DRMs. In order to identify the structural mechanisms of selectivity, we developed a peptide-docking algorithm to predict the atomic structure of protease-substrate complexes and applied it to a large and diverse set of cleavable and noncleavable peptides. Cleavable peptides showed significantly lower energies of interaction than noncleavable peptides with six protease active-site residues playing the most significant role in discrimination. Surprisingly, all six residues correspond to sequence positions associated with drug resistance mutations, demonstrating that the very residues that are responsible for native substrate specificity in HIV-1 protease are altered during its evolution to drug resistance, suggesting that drug resistance and substrate selectivity may share common mechanisms.

Structure and Mechanism of the Lincosamide Antibiotic Adenylyltransferase LinB by Mariya Morar; Kirandeep Bhullar; Donald W. Hughes; Murray Junop; Gerard D. Wright (1649-1659).
Lincosamides make up an important class of antibiotics used against a wide range of pathogens, including methicillin-resistant Staphylococcus aureus. Predictably, lincosamide-resistant microorganisms have emerged with antibiotic modification as one of their major resistance strategies. Inactivating enzymes LinB/A catalyze adenylylation of the drug; however, little is known about their mechanistic and structural properties. We determined two X-ray structures of LinB: ternary substrate– and binary product–bound complexes. Structural and kinetic characterization of LinB, mutagenesis, solvent isotope effect, and product inhibition studies are consistent with a mechanism involving direct in-line nucleotidyl transfer. The characterization of LinB enabled its classification as a member of a nucleotidyltransferase superfamily, along with nucleotide polymerases and aminoglycoside nucleotidyltransferases, and this relationship offers further support for the LinB mechanism. The LinB structure provides an evolutionary link to ancient nucleotide polymerases and suggests that, like protein kinases and acetyltransferases, these are proto-resistance elements from which drug resistance can evolve.

A Role for a Specific Cholesterol Interaction in Stabilizing the Apo Configuration of the Human A2A Adenosine Receptor by Edward Lyman; Chris Higgs; Byungchan Kim; Dmitry Lupyan; John C. Shelley; Ramy Farid; Gregory A. Voth (1660-1668).
The function of G-protein-coupled receptors is tightly modulated by the lipid environment. Long-timescale molecular dynamics simulations (totaling ∼3 μs) of the A2A receptor in cholesterol-free bilayers, with and without the antagonist ZM241385 bound, demonstrate the instability of helix II in the apo receptor in cholesterol-poor membrane regions. We directly observe that the effect of cholesterol binding is to stabilize helix II against a buckling-type deformation, perhaps rationalizing the observation that the A2A receptor couples to G protein only in the presence of cholesterol (). The results suggest a mechanism by which the A2A receptor may function as a coincidence detector, activating only in the presence of both cholesterol and agonist. We also observed a previously hypothesized conformation of the tryptophan “rotameric switch” on helix VI in which a phenylalanine on helix V positions the tryptophan out of the ligand binding pocket.

Human coagulation factor IX serves both to maintain and to control blood coagulation. The dual function of this hemophilic factor is implemented by a tiered activation mechanism. Processed two-chain factor IXa is catalytically silent; only together with its cofactor VIIIa does factor IXa form the highly potent Xase complex. The detailed mechanism of this secondary activation has remained elusive so far. Here we present the crystal structures of Xase-like factor IXa mutants with several-thousand-fold activity enhancement that mimic the secondary activation by Xase formation. The structures reveal how cofactor-triggered and substrate-assisted modulations in the factor IXa 99- and 60-loops cooperate in S4 through S2′ formation, allowing for productive substrate recognition. We could further physically map and visualize a distinct communication line, along which agonists such as Ca2+ direct their effects to the active site and vice versa.

Structural Plasticity of Eph-Receptor A4 Facilitates Cross-Class Ephrin Signaling by Thomas A. Bowden; A. Radu Aricescu; Joanne E. Nettleship; Christian Siebold; Nahid Rahman-Huq; Raymond J. Owens; David I. Stuart; E. Yvonne Jones (1679).

Preparation of Multimilligram Quantities of Large, Linear DNA Molecules for Structural Studies by Merlind Muecke; Martin Samuels; Megan Davey; David Jeruzalmi (1679).