Using Soft X-Rays for a Detailed Picture of Divalent Metal Binding in the Nucleosome

Divalent metals associate with DNA in a site-selective manner, which can influence nucleosome positioning, mobility, compaction, and recognition by nuclear factors. We previously characterized divalent metal binding in the nucleosome core using hard (short-wavelength) X-rays allowing high-resolution crystallographic determination of the strongest affinity sites, which revealed that Mn²⁺ associates with the DNA major groove in a sequence- and conformation-dependent manner. In this study, we obtained diffraction data with soft X-rays at the Mn²⁺ absorption edge for a core particle crystal in the presence of 10 mM MnSO₄, mimicking prevailing Mg²⁺ concentration in the nucleus. This provides an exceptional view of counterion binding in the nucleosome through identification of 45 divalent metal binding sites.In addition to that at the well-characterized major interparticle interface, only one other histone-divalent metal binding site is found, which corresponds to a symmetry-related counterpart on the ‘free’ H2B α1 helix C-terminus. This emphasizes the importance of the α-helix dipole in ion binding and suggests that the H2B motif may serve as a nucleation site in nucleosome compaction. The 43 sites associated with the DNA are characterized by (1) high-affinity direct coordination at the most electrostatically favorable major groove locations, (2) metal hydrate binding to the major groove, (3) direct coordination to phosphate groups at sites of high charge density, (4) metal hydrate binding in the minor groove, or (5) metal hydrate-divalent anion pairing. Metal hydrates are found within the minor groove only at locations displaying a narrow range of high-intermediate width and to which histone N-terminal tails are not associated or proximal. This indicates that divalent metals and histone tails can both collaborate and compete in minor groove association, which sheds light on nucleosome solubility and chromatin compaction behavior.     

Structures of the class D carbapenemase OXA-24 from Acinetobacter baumannii in complex with doripenem

The emergence of class D β-lactamases with carbapenemase activity presents an enormous challenge to health practitioners, particularly with regard to the treatment of infections caused by Gram-negative pathogens such as Acinetobacter baumannii. Unfortunately, class D β-lactamases with carbapenemase activity are resistant to β-lactamase inhibitors. To better understand the details of the how these enzymes bind and hydrolyze carbapenems, we have determined the structures of two deacylation-deficient variants (K84D and V130D) of the class D carbapenemase OXA-24 with doripenem bound as a covalent acyl-enzyme intermediate. Doripenem adopts essentially the same configuration in both OXA-24 variant structures, but varies significantly when compared to the non-carbapenemase class D member OXA-1/doripenem complex. The alcohol of the 6α hydroxyethyl moiety is directed away from the general base carboxy-K84, with implications for activation of the deacylating water. The tunnel formed by the Y112/M223 bridge in the apo form of OXA-24 is largely unchanged by the binding of doripenem. The presence of this bridge, however, causes the distal pyrrolidine/sulfonamide group to bind in a drastically different conformation compared to doripenem bound to OXA-1. The resulting difference in the position of the side-chain bridge sulfur of doripenem is consistent with the hypothesis that the tautomeric state of the pyrroline ring contributes to the different carbapenem hydrolysis rates of OXA-1 and OXA-24. These findings represent a snapshot of a key step in the catalytic mechanism of an important class D enzyme, and might be useful for the design of novel inhibitors.     

A Structural Portrait of the PDZ Domain Family

PDZ (PSD-95/Discs-large/ZO1) domains are interaction modules that typically bind to specific C-terminal sequences of partner proteins and assemble signaling complexes in multicellular organisms. We have analyzed the existing database of PDZ domain structures in the context of a specificity tree based on binding specificities defined by peptide-phage binding selections. We have identified 16 structures of PDZ domains in complex with high-affinity ligands and have elucidated four additional structures to assemble a structural database that covers most of the branches of the PDZ specificity tree. A detailed comparison of the structures reveals features that are responsible for the diverse specificities across the PDZ domain family. Specificity differences can be explained by differences in PDZ residues that are in contact with the peptide ligands, but these contacts involve both side-chain and main-chain interactions. Most PDZ domains bind peptides in a canonical conformation in which the ligand main chain adopts an extended β-strand conformation by interacting in an antiparallel fashion with a PDZ β-strand. However, a subset of PDZ domains bind peptides with a bent main-chain conformation and the specificities of these non-canonical domains could not be explained based on canonical structures. Our analysis provides a structural portrait of the PDZ domain family, which serves as a guide in understanding the structural basis for the diverse specificities across the family.     

The role of RNA sequence and structure in RNA--protein interactions

We investigate the sequence and structural properties of RNA-protein interaction sites in 211 RNA-protein chain pairs, the largest set of RNA-protein complexes analyzed to date. Statistical analysis confirms and extends earlier analyses made on smaller data sets. There are 24.6% of hydrogen bonds between RNA and protein that are nucleobase specific, indicating the importance of both nucleobase-specific and -nonspecific interactions. While there is no significant difference between RNA base frequencies in protein-binding and non-binding regions, distinct preferences for RNA bases, RNA structural states, protein residues, and protein secondary structure emerge when nucleobase-specific and -nonspecific interactions are considered separately. Guanine nucleobase and unpaired RNA structural states are significantly preferred in nucleobase-specific interactions; however, nonspecific interactions disfavor guanine, while still favoring unpaired RNA structural states. The opposite preferences of nucleobase-specific and -nonspecific interactions for guanine may explain discrepancies between earlier studies with regard to base preferences in RNA-protein interaction regions. Preferences for amino acid residues differ significantly between nucleobase-specific and -nonspecific interactions, with nonspecific interactions showing the expected bias towards positively charged residues. Irregular protein structures are strongly favored in interactions with the protein backbone, whereas there is little preference for specific protein secondary structure in either nucleobase-specific interaction or -nonspecific interaction. Overall, this study shows strong preferences for both RNA bases and RNA structural states in protein-RNA interactions, indicating their mutual importance in protein recognition.     

Structural Analysis of Peroxide-Soaked MnSOD Crystals Reveals Side-On Binding of Peroxide to Active-Site Manganese

The superoxide dismutase (SOD) enzymes are important antioxidant agents that protect cells from reactive oxygen species. The SOD family is responsible for catalyzing the disproportionation of superoxide radical to oxygen and hydrogen peroxide. Manganese- and iron-containing SOD exhibit product inhibition whereas Cu/ZnSOD does not. Here, we report the crystal structure of Escherichia coli MnSOD with hydrogen peroxide cryotrapped in the active site. Crystallographic refinement to 1.55 Å and close inspection revealed electron density for hydrogen peroxide in three of the four active sites in the asymmetric unit. The hydrogen peroxide molecules are in the position opposite His26 that is normally assumed by water in the trigonal bipyramidal resting state of the enzyme. Hydrogen peroxide is present in active sites B, C, and D and is side-on coordinated to the active-site manganese. In chains B and D, the peroxide is oriented in the plane formed by manganese and ligands Asp167 and His26. In chain C, the peroxide is bound, making a 70° angle to the plane. Comparison of the peroxide-bound active site with the hydroxide-bound octahedral form shows a shifting of residue Tyr34 towards the active site when peroxide is bound. Comparison with peroxide-soaked Cu/ZnSOD indicates end-on binding of peroxide when the SOD does not exhibit inhibition by peroxide and side-on binding of peroxide in the product-inhibited state of MnSOD.     

On the Structure of the Proton-Binding Site in the Fo Rotor of Chloroplast ATP Synthases

The recently reported crystal structures of the membrane-embedded proton-dependent c-ring rotors of a cyanobacterial F₁F_o ATP synthase and a chloroplast F₁F_o ATP synthase have provided new insights into the mechanism of this essential enzyme. While the overall features of these c-rings are similar, a discrepancy in the structure and hydrogen-bonding interaction network of the H⁺ sites suggests two distinct binding modes, potentially reflecting a mechanistic differentiation. Importantly, the conformation of the key glutamate side chain to which the proton binds is also altered. To investigate the nature of these differences, we use molecular dynamics simulations of both c-rings embedded in a phospholipid membrane. We observe that the structure of the c₁₅ ring from Spirulina platensis is unequivocally stable within the simulation time. By contrast, the proposed structure of the H⁺ site in the chloroplast c₁₄ ring changes rapidly and consistently into that reported for the c₁₅ ring, indicating that the latter represents a common binding mode. To assess this hypothesis, we have remodeled the c₁₄ ring by molecular replacement using the published structure factors. The resulting structure provides clear evidence in support of a common binding site conformation and is also considerably improved statistically. These findings, taken together with a sequence analysis of c-subunits in the ATP synthase family, indicate that the so-called proton-locked conformation observed in the c₁₅ ring may be a common characteristic not only of light-driven systems such as chloroplasts and cyanobacteria but also of a selection of other bacterial species.     

Mapping of protein-protein interaction sites by the 'absence of interference' approach

Protein-protein interactions are critical to most biological processes, and locating protein-protein interfaces on protein structures is an important task in molecular biology. We developed a new experimental strategy called the ‘absence of interference’ approach to determine surface residues involved in protein-protein interaction of established yeast two-hybrid pairs of interacting proteins. One of the proteins is subjected to high-level randomization by error-prone PCR. The resulting library is selected by yeast two-hybrid system for interacting clones that are isolated and sequenced. The interaction region can be identified by an absence or depletion of mutations. For data analysis and presentation, we developed a Web interface that analyzes the mutational spectrum and displays the mutational frequency on the surface of the structure (or a structural model) of the randomized protein†. Additionally, this interface might be of use for the display of mutational distributions determined by other types of random mutagenesis experiments. We applied the approach to map the interface of the catalytic domain of the DNA methyltransferase Dnmt3a with its regulatory factor Dnmt3L. Dnmt3a was randomized with high mutational load. A total of 76 interacting clones were isolated and sequenced, and 648 mutations were identified. The mutational pattern allowed to identify a unique interaction region on the surface of Dnmt3a, which comprises about 500-600 Å². The results were confirmed by site-directed mutagenesis and structural analysis. The absence-of-interference approach will allow high-throughput mapping of protein interaction sites suitable for functional studies and protein docking.     

Analysis of Four-Way Junctions in RNA Structures

RNA secondary structures can be divided into helical regions composed of canonical Watson-Crick and related base pairs, as well as single-stranded regions such as hairpin loops, internal loops, and junctions. These elements function as building blocks in the design of diverse RNA molecules with various fundamental functions in the cell. To better understand the intricate architecture of three-dimensional (3D) RNAs, we analyze existing RNA four-way junctions in terms of base-pair interactions and 3D configurations. Specifically, we identify nine broad junction families according to coaxial stacking patterns and helical configurations. We find that helices within junctions tend to arrange in roughly parallel and perpendicular patterns and stabilize their conformations using common tertiary motifs such as coaxial stacking, loop-helix interaction, and helix packing interaction. Our analysis also reveals a number of highly conserved base-pair interaction patterns and novel tertiary motifs such as A-minor-coaxial stacking combinations and sarcin/ricin motif variants. Such analyses of RNA building blocks can ultimately help in the difficult task of RNA 3D structure prediction.     

A Survey of λ Repressor Fragments from Two-State to Downhill Folding

We survey the two-state to downhill folding transition by examining 20 λ_6-85^? mutants that cover a wide range of stabilities and folding rates. We investigated four new λ_6-85^? mutants designed to fold especially rapidly. Two were engineered using the core remodeling of Lim and Sauer, and two were engineered using Ferreiro et al.'s frustratometer. These proteins have probe-dependent melting temperatures as high as 80 °C and exhibit a fast molecular phase with the characteristic temperature dependence of the amplitude expected for downhill folding. The survey reveals a correlation between melting temperature and downhill folding previously observed for the β-sheet protein WW domain. A simple model explains this correlation and predicts the melting temperature at which downhill folding becomes possible. An X-ray crystal structure with a 1.64-Å resolution of a fast-folding mutant fragment shows regions of enhanced rigidity compared to the full wild-type protein.     

Crystal Structures of Bacillus subtilis Lon Protease

Lon ATP-dependent proteases are key components of the protein quality control systems of bacterial cells and eukaryotic organelles. Eubacterial Lon proteases contain an N-terminal domain, an ATPase domain, and a protease domain, all in one polypeptide chain. The N-terminal domain is thought to be involved in substrate recognition, the ATPase domain in substrate unfolding and translocation into the protease chamber, and the protease domain in the hydrolysis of polypeptides into small peptide fragments. Like other AAA+ ATPases and self-compartmentalising proteases, Lon functions as an oligomeric complex, although the subunit stoichiometry is currently unclear. Here, we present crystal structures of truncated versions of Lon protease from Bacillus subtilis (BsLon), which reveal previously unknown architectural features of Lon complexes. Our analytical ultracentrifugation and electron microscopy show different oligomerisation of Lon proteases from two different bacterial species, Aquifex aeolicus and B. subtilis. The structure of BsLon-AP shows a hexameric complex consisting of a small part of the N-terminal domain, the ATPase, and protease domains. The structure shows the approximate arrangement of the three functional domains of Lon. It also reveals a resemblance between the architecture of Lon proteases and the bacterial proteasome-like protease HslUV. Our second structure, BsLon-N, represents the first 209 amino acids of the N-terminal domain of BsLon and consists of a globular domain, similar in structure to the E. coli Lon N-terminal domain, and an additional four-helix bundle, which is part of a predicted coiled-coil region. An unexpected dimeric interaction between BsLon-N monomers reveals the possibility that Lon complexes may be stabilised by coiled-coil interactions between neighbouring N-terminal domains. Together, BsLon-N and BsLon-AP are 36 amino acids short of offering a complete picture of a full-length Lon protease.     

Systematic Mutational Analysis of Peptide Inhibition of the p53-MDM2/MDMX Interactions

Inhibition of the interaction between the tumor suppressor protein p53 and its negative regulators MDM2 and MDMX is of great interest in cancer biology and drug design. We previously reported a potent duodecimal peptide inhibitor, termed PMI (TSFAEYWNLLSP), of the p53-MDM2 and -MDMX interactions. PMI competes with p53 for MDM2 and MDMX binding at an affinity roughly 2 orders of magnitude higher than that of ^17-28p53 (ETFSDLWKLLPE) of the same length; both peptides adopt nearly identical α-helical conformations in the complexes, where the three highlighted hydrophobic residues Phe, Trp, and Leu dominate PMI or ^17-28p53 binding to MDM2 and MDMX. To elucidate the molecular determinants for PMI activity and specificity, we performed a systematic Ala scanning mutational analysis of PMI and ^17-28p53. The binding affinities for MDM2 and MDMX of a total of 35 peptides including 10 truncation analogs were quantified, affording a complete dissection of energetic contributions of individual residues of PMI and ^17-28p53 to MDM2 and MDMX association. Importantly, the N8A mutation turned PMI into the most potent dual-specific antagonist of MDM2 and MDMX reported to date, registering respective K_d values of 490 pM and 2.4 nM. The co-crystal structure of N8A-PMI-^25-109MDM2 was determined at 1.95 Å, affirming that high-affinity peptide binding to MDM2/MDMX necessitates, in addition to optimized intermolecular interactions, enhanced helix stability or propensity contributed by non-contact residues. The powerful empirical binding data and crystal structures present a unique opportunity for computational studies of peptide inhibition of the p53-MDM2/MDMX interactions.     

Crystal Structures and Enzyme Mechanisms of a Dual Fucose Mutarotase/Ribose Pyranase

Escherichia coli FucU (Fucose Unknown) is a dual fucose mutarotase and ribose pyranase, which shares 44% sequence identity with its human counterpart. Herein, we report the structures of E. coli FucU and mouse FucU bound to l-fucose and delineate the catalytic mechanisms underlying the interconversion between stereoisomers of fucose and ribose. E. coli FucU forms a decameric toroid with each active site formed by two adjacent subunits. While one subunit provides most of the fucose-interacting residues including a catalytic tyrosine residue, the other subunit provides a catalytic His-Asp dyad. This active-site feature is critical not only for the mutarotase activity toward l-fucose but also for the pyranase activity toward d-ribose. Structural and biochemical analyses pointed that mouse FucU assembles into four different oligomeric forms, among which the smallest homodimeric form is most abundant and would be the predominant species under physiological conditions. This homodimer has two fucose-binding sites that are devoid of the His-Asp dyad and catalytically inactive, indicating that the mutarotase and the pyranase activities appear dispensable in vertebrates. The defective assembly of the mouse FucU homodimer into the decameric form is due to an insertion of two residues at the N-terminal extreme, which is a common aspect of all the known vertebrate FucU proteins. Therefore, vertebrate FucU appears to serve for as yet unknown function through the quaternary structural alteration.     

A new classification of membrane protein crystals

Although being much smaller than the number of soluble proteins in the Protein Data Bank, the number of membrane proteins therein now approaches 700, and a statistical analysis becomes meaningful. Such an analysis showed that the conventional subdivision into monotopic, β-barrel and α-helical membrane proteins is appropriate but should be amended by a classification according to the detergent micelle structure in the crystal, which can be derived from the packing of the membrane-immersed parts of the proteins. The crystal packing density is specific for the three conventional types of membrane proteins and soluble proteins. It is also specific for three observed detergent arrangements that are micelle pockets, micelle filaments and micelle sheets, demonstrating that the detergent structure affects crystallization. The packing density distribution of crystals from integral membrane proteins has approximately the same shape as that of soluble proteins but is by a factor of two broader and shifted to lower density. It seems unlikely that the differences can be explained by a mere solvent expansion due to the required detergent. The crystallized membrane proteins were further analyzed with respect to protein mass, oligomerization and crystallographic asymmetric unit, space group, crystal ordering and symmetry. The results provide a new view on membrane proteins.     

Structural Basis of Conserved Cysteine in the Fibroblast Growth Factor Family: Evidence for a Vestigial Half-Cystine

The 22 members of the mouse/human fibroblast growth factor (FGF) family of proteins contain a conserved cysteine residue at position 83 (numbering scheme of the 140-residue form of FGF-1). Sequence and structure information suggests that this position is a free cysteine in 16 members and participates as a half-cystine in at least 3 (and perhaps as many as 6) other members. While a structural role as a half-cystine provides a stability basis for possible selective pressure, it is less clear why this residue is conserved as a free cysteine (although free buried thiols can limit protein functional half-life). To probe the structural role of the free cysteine at position 83 in FGF-1, we constructed Ala, Ser, Thr, Val, and Ile mutations and determined their effects on structure and stability. These results show that position 83 in FGF-1 is thermodynamically optimized to accept a free cysteine. A second cysteine mutation was introduced into wild-type FGF-1 at adjacent position Ala66, which is known to participate as a half-cystine with position 83 in FGF-8, FGF-19, and FGF-23. Results show that, unlike position 83, a free cysteine at position 66 destabilizes FGF-1; however, upon oxidation, a near-optimal disulfide bond is formed between Cys66 and Cys83, resulting in ∼ 14 kJ/mol of increased thermostability. Thus, while the conserved free cysteine at position 83 in the majority of the FGF proteins may have a principal role in limiting functional half-life, evidence suggests that it is a vestigial half-cystine.