The crystal structure of the rare-cutting restriction enzyme SdaI reveals unexpected domain architecture

Rare-cutting restriction enzymes are important tools in genome analysis. We report here the crystal structure of SdaI restriction endonuclease, which is specific for the 8 bp sequence CCTGCA/GG ("/" designates the cleavage site). Unlike orthodox Type IIP enzymes, which are single domain proteins, the SdaI monomer is composed of two structural domains. The N domain contains a classical winged helix-turn-helix (wHTH) DNA binding motif, while the C domain shows a typical restriction endonuclease fold. The active site of SdaI is located within the C domain and represents a variant of the canonical PD-(D/E)XK motif. SdaI determinants of sequence specificity are clustered on the recognition helix of the wHTH motif at the N domain. The modular architecture of SdaI, wherein one domain mediates DNA binding while the other domain is predicted to catalyze hydrolysis, distinguishes SdaI from previously characterized restriction enzymes interacting with symmetric recognition sequences.     

Molecular Analysis of pSK1 par: A Novel Plasmid Partitioning System Encoded by Staphylococcal Multiresistance Plasmids

The segregation of prokaryotic plasmids typically requires a centromere-like site and two proteins, a centromere-binding protein (CBP) and an NTPase. By contrast, a single 245 residue Par protein mediates partition of the prototypical staphylococcal multiresistance plasmid pSK1 in the absence of an identifiable NTPase component. To gain insight into centromere binding by pSK1 Par and its segregation function we performed structural, biochemical and in vivo studies. Here we show that pSK1 Par binds a centromere consisting of seven repeat elements. We demonstrate this Par-centromere interaction also mediates Par autoregulation. To elucidate the Par centromere binding mechanism, we obtained a structure of the Par N-terminal DNA-binding domain bound to centromere DNA to 2.25 Å. The pSK1 Par structure, which harbors a winged-helix-turn-helix (wHTH), is distinct from other plasmid CBP structures but shows homology to the B. subtilis chromosome segregation protein, RacA. Biochemical studies suggest the region C-terminal to the Par wHTH forms coiled coils and mediates oligomerization. Fluorescence microscopy analyses show that pSK1 Par enhances the separation of plasmids from clusters, driving effective segregation upon cell division. Combined the data provide insight into the molecular properties of a single protein partition system.     

A family portrait of the RIO kinases

The RIO family of atypical serine protein kinases has been first characterized only recently. It consists of enzymes that contain a unique domain with a characteristic kinase sequence motif and usually some additional domains. At least two RIO proteins, Rio1 and Rio2, are present in organisms varying from Archaea to humans, with a third Rio3 subfamily present only in multicellular eukaryotes. Yeast Rio1 and Rio2 proteins have been implicated in the processing of 20 S pre-rRNA and are necessary for survival of the cells. Crystal structures of Archaeoglobus fulgidus Rio1 and Rio2 have shown that whereas the overall fold of these enzymes resembles typical protein kinases, some of the structural domains, particularly those involved in peptide substrate binding, are not present. The mode of binding of nucleotides also differs from that found in typical protein kinases. Although it has been shown that both Rio1 and Rio2 have the enzymatic activity of kinases and are capable of autophosphorylation, the biological substrates of RIO proteins and their full biological role still remain to be discovered.     

Structural analysis of the lymphocyte-specific kinase Lck in complex with non-selective and Src family selective kinase inhibitors.

BACKGROUND: The lymphocyte-specific kinase Lck is a member of the Src family of non-receptor tyrosine kinases. Lck catalyzes the initial phosphorylation of T-cell receptor components that is necessary for signal transduction and T-cell activation. On the basis of both biochemical and genetic studies, Lck is considered an attractive cell-specific target for the design of novel T-cell immunosuppressants. To date, the lack of detailed structural information on the mode of inhibitor binding to Lck has limited the discovery of novel Lck inhibitors. RESULTS: We report here the high-resolution crystal structures of an activated Lck kinase domain in complex with three structurally distinct ATP-competitive inhibitors: AMP-PNP (a non-selective, non-hydrolyzable ATP analog); staurosporine (a potent but non-selective protein kinase inhibitor); and PP2 (a potent Src family selective protein tyrosine kinase inhibitor). Comparison of these structures reveals subtle but important structural changes at the ATP-binding site. Furthermore, PP2 is found to access a deep, hydrophobic pocket near the ATP-binding cleft of the enzyme; this binding pocket is not occupied by either AMP-PNP or staurosporine. CONCLUSIONS: The potency of staurosporine against Lck derives in part from an induced movement of the glycine-rich loop of the enzyme upon binding of this ligand, which maximizes the van der Waals interactions present in the complex. In contrast, PP2 binds tightly and selectively to Lck and other Src family kinases by making additional contacts in a deep, hydrophobic pocket adjacent to the ATP-binding site; the amino acid composition of this pocket is unique to Src family kinases. The structures of these Lck complexes offer useful structural insights as they demonstrate that kinase selectivity can be achieved with small-molecule inhibitors that exploit subtle topological differences among protein kinases.     

Dominant Rio1 kinase/ATPase catalytic mutant induces trapping of late pre-40S biogenesis factors in 80S-like ribosomes

During eukaryotic ribosome biogenesis, members of the conserved atypical serine/threonine protein kinase family, the RIO kinases (Rio1, Rio2 and Rio3) function in small ribosomal subunit biogenesis. Structural analysis of Rio2 indicated a role as a conformation-sensing ATPase rather than a kinase to regulate its dynamic association with the pre-40S subunit. However, it remained elusive at which step and by which mechanism the other RIO kinase members act. Here, we have determined the crystal structure of the human Rio1–ATP–Mg²⁺ complex carrying a phosphoaspartate in the active site indicative of ATPase activity. Structure-based mutations in yeast showed that Rio1''s catalytic activity regulates its pre-40S association. Furthermore, we provide evidence that Rio1 associates with a very late pre-40S via its conserved C-terminal domain. Moreover, a rio1 dominant-negative mutant defective in ATP hydrolysis induced trapping of late biogenesis factors in pre-ribosomal particles, which turned out not to be pre-40S but 80S-like ribosomes. Thus, the RIO kinase fold generates a versatile ATPase enzyme, which in the case of Rio1 is activated following the Rio2 step to regulate one of the final 40S maturation events, at which time the 60S subunit is recruited for final quality control check.     

Insights into specific DNA recognition during the assembly of a viral genome packaging machine

Terminase enzymes mediate genome "packaging" during the reproduction of DNA viruses. In lambda, the gpNu1 subunit guides site-specific assembly of terminase onto DNA. The structure of the dimeric DNA binding domain of gpNu1 was solved using nuclear magnetic resonance spectroscopy. Its fold contains a unique winged helix-turn-helix (wHTH) motif within a novel scaffold. Surprisingly, a predicted P loop ATP binding motif is in fact the wing of the DNA binding motif. Structural and genetic analysis has identified determinants of DNA recognition specificity within the wHTH motif and the DNA recognition sequence. The structure reveals an unexpected DNA binding mode and provides a mechanistic basis for the concerted action of gpNu1 and Escherichia coli integration host factor during assembly of the packaging machinery.     

Structural and Biophysical Characterization of the Syk Activation Switch

Syk is an essential non-receptor tyrosine kinase in intracellular immunological signaling, and the control of Syk kinase function is considered as a valuable target for pharmacological intervention in autoimmune or inflammation diseases. Upon immune receptor stimulation, the kinase activity of Syk is regulated by binding of phosphorylated immune receptor tyrosine-based activating motifs (pITAMs) to the N-terminal tandem Src homology 2 (tSH2) domain and by autophosphorylation with consequences for the molecular structure of the Syk protein. Here, we present the first crystal structures of full-length Syk (fl-Syk) as wild type and as Y348F,Y352F mutant forms in complex with AMP-PNP revealing an autoinhibited conformation. The comparison with the crystal structure of the truncated Syk kinase domain in complex with AMP-PNP taken together with ligand binding studies by surface plasmon resonance (SPR) suggests conformational differences in the ATP sites of autoinhibited and activated Syk forms. This hypothesis was corroborated by studying the thermodynamic and kinetic interaction of three published Syk inhibitors with isothermal titration calorimetry and SPR, respectively. We further demonstrate the modulation of inhibitor binding affinities in the presence of pITAM and discuss the observed differences of thermodynamic and kinetic signatures. The functional relevance of pITAM binding to fl-Syk was confirmed by a strong stimulation of in vitro autophosphorylation. A structural feedback mechanism on the kinase domain upon pITAM binding to the tSH2 domain is discussed in analogy of the related family kinase ZAP-70 (Zeta-chain-associated protein kinase 70). Surprisingly, we observed distinct conformations of the tSH2 domain and the activation switch including Tyr348 and Tyr352 in the interdomain linker of Syk in comparison to ZAP-70.     

Insights into the dynamic interactions at chemokine-receptor interfaces and mechanistic models of chemokine binding

Chemokine receptors are the central signaling hubs of several processes such as cell migration, chemotaxis and cell positioning. In this graphical review, we provide an overview of the structural and mechanistic principles governing chemokine recognition that are currently emerging. Structural models of chemokine-receptor co-complexes with endogenous chemokines, viral chemokines and therapeutics have been resolved that highlight multiple interaction sites, termed as CRS1, CRS1.5 etc. The first site of interaction has been shown to be the N-terminal domain of the receptors (CRS1 site). A large structural flexibility of the N-terminal domain has been reported that was supported by both experimental and simulation studies. Upon chemokine binding, the N-terminal domain appears to show constricted dynamics and opens up to interact with the chemokine via a large interface. The subsequent sites such as CRS1.5 and CRS2 sites have been structurally well resolved although differences arise such as the localization of the N-terminus of the ligand to a major or minor pocket of the orthosteric binding site. Several computational studies have highlighted the dynamic protein-protein interface at the CRS1 site that seemingly appears to resolve the differences in NMR and mutagenesis studies. Interestingly, the differential dynamics at the CRS1 site suggests a mixed model of binding with complex signatures of both conformational selection and induced fit models. Integrative experimental and computational approaches could help unravel the structural basis of promiscuity and specificity in chemokine-receptor binding and open up new avenues of therapeutic design.     

Atypical (RIO) protein kinases from Haemonchus contortus--promise as new targets for nematocidal drugs

Almost nothing is known about atypical kinases in multicellular organisms, including parasites. Supported by information and data available for the free-living nematode, Caenorhabditis elegans, and other eukaryotes, the present article describes three RIO kinase genes, riok-1, riok-2 and riok-3, from Haemonchus contortus, one of the most important parasitic nematodes of small ruminants. Analyses of these genes and their products predict that they each play critical roles in the developmental pathways of parasitic nematodes. The findings of this review indicate prospects for functional studies of these genes in C. elegans (as a surrogate) and opportunities for the design of a novel class of nematode-specific inhibitors of RIO kinases. The latter aspect is of paramount importance, given the serious problems linked to anthelmintic resistance in parasitic nematode populations of livestock.     

Structural and functional insights into the molecular mechanisms responsible for the regulation of pyruvate dehydrogenase kinase 2

PDHK2 is a mitochondrial protein kinase that phosphorylates pyruvate dehydrogenase complex, thereby down-regulating the oxidation of pyruvate. Here, we present the crystal structure of PDHK2 bound to the inner lipoyl-bearing domain of dihydrolipoamide transacetylase (L2) determined with or without bound adenylyl imidodiphosphate. Both structures reveal a PDHK2 dimer complexed with two L2 domains. Comparison with apo-PDHK2 shows that L2 binding causes rearrangements in PDHK2 structure that affect the L2- and E1-binding sites. Significant differences are found between PDHK2 and PDHK3 with respect to the structure of their lipoyllysine-binding cavities, providing the first structural support to a number of studies showing that these isozymes are markedly different with respect to their affinity for the L2 domain. Both structures display a novel type II potassium-binding site located on the PDHK2 interface with the L2 domain. Binding of potassium ion at this site rigidifies the interface and appears to be critical in determining the strength of L2 binding. Evidence is also presented that potassium ions are indispensable for the cross-talk between the nucleotide- and L2-binding sites of PDHK2. The latter is believed to be essential for the movement of PDHK2 along the surface of the transacetylase scaffold.     

NMR Structure of the Amino-Terminal Domain of the Lambda Integrase Protein in Complex with DNA: Immobilization of a Flexible Tail Facilitates Beta-Sheet Recognition of the Major Groove

The integrase protein (Int) from bacteriophage lambda is the archetypal member of the tyrosine recombinase family, a large group of enzymes that rearrange DNA in all domains of life. Int catalyzes the insertion and excision of the viral genome into and out of the Escherichia coli chromosome. Recombination transpires within higher-order nucleoprotein complexes that form when its amino-terminal domain binds to arm-type DNA sequences that are located distal to the site of strand exchange. Arm-site binding by Int is essential for catalysis, as it promotes Int-mediated bridge structures that stabilize the recombination machinery. We have elucidated how Int is able to sequence specifically recognize the arm-type site sequence by determining the solution structure of its amino-terminal domain (Int^N, residues Met1 to Leu64) in complex with its P′2 DNA binding site. Previous studies have shown that Int^N adopts a rare monomeric DNA binding fold that consists of a three-stranded antiparallel beta-sheet that is packed against a carboxy-terminal alpha helix. A low-resolution crystal structure of the full-length protein also revealed that the sheet is inserted into the major groove of the arm-type site. The solution structure presented here reveals how Int^N specifically recognizes the arm-type site sequence. A novel feature of the new solution structure is the use of an 11-residue tail that is located at the amino terminus. DNA binding induces the folding of a 3₁₀ helix in the tail that projects the amino terminus of the protein deep into the minor groove for stabilizing DNA contacts. This finding reveals the structural basis for the observation that the “unstructured” amino terminus is required for recombination.     

Identification of one of the apurinic/apyrimidinic lyase active sites of topoisomerase V by structural and functional studies

Topoisomerase V (Topo-V) is the only member of a novel topoisomerase subtype. Topo-V is unique because it is a bifunctional enzyme carrying both topoisomerase and DNA repair lyase activities within the same protein. Previous studies had shown that the topoisomerase domain spans the N-terminus of the protein and is followed by 12 tandem helix–hairpin–helix [(HhH)₂] domains. There are at least two DNA repair lyase active sites for apurinic/apyrimidinic (AP) site processing, one within the N-terminal region and the second within the C-terminal domain of Topo-V, but their exact locations and characteristics are unknown. In the present study, the N-terminal 78-kDa fragment of Topo-V (Topo-78), containing the topoisomerase domain and one of the lyase DNA repair domains, was characterized by structural and biochemical studies. The results show that an N-terminal 69-kDa fragment is the minimal fragment with both topoisomerase and AP lyase activities. The lyase active site of Topo-78 is at the junction of the fifth and sixth (HhH)₂ domains. From the biochemical and structural data, it appears that Lys571 is the most probable nucleophile responsible for the lyase activity. Our experiments also suggest that Topo-V most likely acts as a Class I AP endonuclease in vivo.