Proteins at atomic resolution

Experimental advances in data collection, including bright sources, cryogenic cooling and two-dimensional detectors, have made it tractable to record data to beyond 1.2 Å for several proteins, yielding high-accuracy models and fine details of structure. For small metalloproteins, atomic-resolution data have enabled ab initio solution of the phase problem.     

The ribosome at atomic resolution

The publication of atomic resolution crystal structures for the large ribosomal subunit from Haloarcula marismortui and the small ribosomal subunit from Thermus thermophilus has permanently altered the way protein synthesis is conceptualized and experiments designed to address its unresolved issues. The impact of these structures on RNA biochemistry is certain to be no less profound. The background and substance of these developments are reviewed here.     

Approaching translation at atomic resolution

Atomic resolution structures of 50S and 30S ribosomal particles have recently been solved by X-ray diffraction. These ribosomal structures show often unusual folds of ribosomal RNAs and proteins, and provide molecular explanations for fundamental aspects of translation. In the 50S structure, the active site for peptide bond formation was localized and found to consist of RNA. The ribosome is thus a ribozyme. In the 30S structures, tRNA binding sites were located, and molecular mechanisms for ribosomal fidelity were proposed. The 30S subunit particle has three globular domains, and relative movements of these domains may be required for translocation of the ribosome during protein synthesis. The structures are consistent with and rationalize decades of biochemical analysis of translation and usher in a molecular age in understanding the ribosome.     

The bacterial ribosome at atomic resolution

X-ray crystallographic structures have just been published for the 30S ribosomal subunit of Thermus thermophilus at 3.4 A resolution and for the 50S subunit of Haloarcula marismortui at 2.4 A. These eagerly awaited structures will provide an enormous boost to research into the mechanisms involved in protein biosynthesis.     

The active principle of garlic at atomic resolution

Despite the fact that many cultures around the world value and utilize garlic as a fundamental component of their cuisine as well as of their medicine cabinets, relatively little is known about the plant's protein configuration that is responsible for the specific properties of garlic. Here, we report the three-dimensional structure of the garlic enzyme alliinase at 1.5 A resolution. Alliinase constitutes the major protein component in garlic bulbs, and it is able to cleave carbon-sulfur bonds. The active enzyme is a pyridoxal-5'-phosphate-dependent homodimeric glycoprotein and belongs to the class I family of pyridoxal-5'-phosphate-dependent enzymes. In addition, it contains a novel epidermal growth factor-like domain that makes it unique among all pyridoxal-5'-phosphate-dependent enzymes.     

Conformational conversion during amyloid formation at atomic resolution

Numerous studies of amyloid assembly have indicated that partially folded protein species are responsible for initiating aggregation. Despite their importance, the structural and dynamic features of amyloidogenic intermediates and the molecular details of how they cause aggregation remain elusive. Here, we use ΔN6, a truncation variant of the naturally amyloidogenic protein β(2)-microglobulin (β(2)m), to determine the solution structure of a nonnative amyloidogenic intermediate at high resolution. The structure of ΔN6 reveals a major repacking of the hydrophobic core to accommodate the nonnative peptidyl-prolyl trans-isomer at Pro32. These structural changes, together with a concomitant pH-dependent enhancement in backbone dynamics on a microsecond-millisecond timescale, give rise to a rare conformer with increased amyloidogenic potential. We further reveal that catalytic amounts of ΔN6 are competent to convert nonamyloidogenic human wild-type β(2)m (Hβ(2)m) into a rare amyloidogenic conformation and provide structural evidence for the mechanism by which this conformational conversion occurs.     

Observation of correlated X-ray scattering at atomic resolution

Tools to study disordered systems with local structural order, such as proteins in solution, remain limited. Such understanding is essential for e.g. rational drug design. Correlated X-ray scattering (CXS) has recently attracted new interest as a way to leverage next-generation light sources to study such disordered matter. The CXS experiment measures angular correlations of the intensity caused by the scattering of X-rays from an ensemble of identical particles, with disordered orientation and position. Averaging over 15 496 snapshot images obtained by exposing a sample of silver nanoparticles in solution to a micro-focused synchrotron radiation beam, we report on experimental efforts to obtain CXS signal from an ensemble in three dimensions. A correlation function was measured at wide angles corresponding to atomic resolution that matches theoretical predictions. These preliminary results suggest that other CXS experiments on disordered ensembles—such as proteins in solution—may be feasible in the future.     

EJCs at the heart of translational control

In mammalian cells, the splicing machinery deposits the exon junction complex (EJC) on mRNA splice junctions. Two studies in this issue now link the EJC to different aspects of translational control. Ma et al. (2008) show that the EJC activates translation downstream of the mTOR signaling pathway, whereas Isken et al. (2008) establish that translation is repressed by partners of the EJC that are implicated in nonsense mediated decay (NMD).     

Molecular structure of cyclic deoxydiadenylic acid at atomic resolution

The molecular structure of a small cyclic nucleotide, cyclic deoxydiadenylic acid, has been determined by single-crystal X-ray diffraction analysis and refined to an R factor of 7.8% at 1.0-A resolution. The crystals are in the monoclinic space group C2 with unit cell dimensions of a = 24.511 (3) A, b = 24.785 (3) A, c = 13.743 (3) A, and beta = 94.02 (2) degrees. The structure was solved by the direct methods program SHELXS-86. There are 2 independent cyclic d(ApAp) molecules, 2 hydrated magnesium ions, and 26 water molecules in the asymmetric unit of the unit cell. The two cyclic d(ApAp) molecules have similar conformations within their 12-membered sugar-phosphate backbone ring, but they have quite different appearances due to the different glycosyl torsion angles that make one molecule more compact and the other extended and open. Three of the four deoxyribose rings are in the less common C3'-endo conformation. All four phosphate groups have their phosphodiester torsion angles alpha/zeta in the gauche(+)/gauche(+) conformation. One of the cyclic d(ApAp) molecules associates with another symmetry-related molecule to form a self-intercalated dimer that is a stable structure in solution, as observed in NMR studies. Many interesting intermolecular interactions, including base-base stacking, ribose-base stacking, base pairing, base-phosphate hydrogen bonding, and metal ion-phosphate interactions, are found in the crystal lattice. This structure may be relevant for understanding the conformational potentiality of an endogenous biological regulator of cellulose synthesis, cyclic (GpGp).     

Characterization at atomic resolution of peptide helical structures.

A survey of literature for the various types of helices experimentally observed in high-resolution single crystal x-ray diffraction analyses of peptides has allowed to determine accurate conformational and helical parameters for the various secondary structures such as the alpha-helix, the 3(10)-helix, the fully extended conformation (2(5)-helix) and the beta-bend ribbon spiral. For each of these structures the characteristic phi, psi conformational parameters, n, the number of residues per turn, h, the height per residues and p, the pitch of the helix are described.     

NMR structure of a parallel-stranded DNA duplex at atomic resolution

DNA dodecamers have been designed with two cytosines on each end and intervening A and T stretches, such that the oligomers have fully complementary A:T base pairs when aligned in the parallel orientation. Spectroscopic (UV, CD and IR), NMR and molecular dynamics studies have shown that oligomers having the sequences d(CCATAATTTACC) and d(CCTATTAAATCC) form a parallel-stranded duplex when dissolved at 1:1 stoichiometry in aqueous solution. This is due to the C:C⁺ clamps on either end and extensive mismatches in the antiparallel orientation. The structure is stable at neutral and acidic pH. At higher temperatures, the duplex melts into single strands in a highly cooperative fashion. All adenine, cytosine and thymine nucleotides adopt the anti conformation with respect to the glycosidic bond. The A:T base pairs form reverse Watson–Crick base pairs. The duplex shows base stacking and NOEs between the base protons T(H6)/A(H8) and the sugar protons (H1′/H2′/H2″) of the preceding nucleotide, as has been observed in antiparallel duplexes. However, no NOEs are observed between base protons H2/H6/H8 of sequential nucleotides, though such NOEs are observed between T(CH₃) and A(H8). A three-dimensional structure of the parallel-stranded duplex at atomic resolution has been obtained using molecular dynamics simulations under NMR constraints. The simulated structures have torsional angles very similar to those found in B-DNA duplexes, but the base stacking and helicoid parameters are significantly different.     

Refined atomic model of glutamine synthetase at 3.5 A resolution

An atomic model of 43,692 non-hydrogen atoms has been determined for the 12-subunit enzyme glutamine synthetase from Salmonella typhimurium, by methods of x-ray diffraction including restrained least-squares atomic refinement against 65,223 unique reflections. At 3.5 A resolution the crystallographic R-factor (on 2 sigma data) is 25.8%. As reported earlier for the unrefined structure, the 12 subunits are arranged in two layers of six; at the interface of pairs of subunits within each layer, cylindrical active sites are formed by six anti-parallel beta strands contributed by one subunit and two strands by the neighboring subunit. This interpretation of the electron density map has now been supported by comparison with glutamine synthetase from Escherichia coli by the Fourier difference method. Each active site cylinder holds two Mn2+ ions, with each ion having as ligands three protein side chains and two water molecules (one water shared by both metals), as well as a histidyl side chain just beyond liganding distance. The protein ligands to Mn2+ 469 are Glu-131, Glu-212, and Glu-220; those to Mn2+ 470 are Glu-129, His-269, and Glu-357. The two layers of subunits are held together largely by the apolar COOH terminus, a helical thong, which inserts into a hydrophobic pocket formed by two neighboring subunits on the opposite ring. Also between layers, there is a hydrogen-bonded beta sheet interaction, as there is between subunits within a ring, but hydrophobic interactions account for most of the intersubunit stability. The central loop, which extends into the central aqueous channel, is subject to attack by at least five enzymes and is discussed as an enzyme "passive site."     

Complete reaction cycle of a cocaine catalytic antibody at atomic resolution

Antibody 7A1 hydrolyzes cocaine to produce nonpsychoactive metabolites ecgonine methyl ester and benzoic acid. Crystal structures of 7A1 Fab' and six complexes with substrate cocaine, the transition state analog, products ecgonine methyl ester and benzoic acid together and individually, as well as heptaethylene glycol have been analyzed at 1.5-2.3 angstroms resolution. Here, we present snapshots of the complete cycle of the cocaine hydrolytic reaction at atomic resolution. Significant structural rearrangements occur along the reaction pathway, but they are generally limited to the binding site, including the ligands themselves. Several interacting side chains either change their rotamers or alter their mobility to accommodate the different reaction steps. CDR loop movements (up to 2.3 angstroms) and substantial side chain rearrangements (up to 9 angstroms) alter the shape and size (approximately 320-500 angstroms3) of the antibody active site from "open" to "closed" to "open" for the substrate, transition state, and product states, respectively.