SAXS and the Working Protein

In this issue of Structure, Davies et al., 2005 present shape reconstructions for the molecular motor p97 using small angle X-ray scattering (SAXS) and offer insights into how ATP consumption is coupled to cyclical domain motions. This work emphasizes the emerging potential of SAXS for visualizing the workings of biological machines in solution

Activation of tyrosine kinases by mutation of the gatekeeper threonine.

Protein kinases targeted by small-molecule inhibitors develop resistance through mutation of the gatekeeper threonine residue of the active site. Here we show that the gatekeeper mutation in the cellular forms of c-ABL, c-SRC, platelet-derived growth factor receptor-alpha and -beta, and epidermal growth factor receptor activates the kinase and promotes malignant transformation of BaF3 cells. Structural analysis reveals that a network of hydrophobic interactions-the hydrophobic spine-characteristic of the active kinase conformation is stabilized by the gatekeeper substitution. Substitution of glycine for the residues constituting the spine disrupts the hydrophobic connectivity and inactivates the kinase. Furthermore, a small-molecule inhibitor that maximizes complementarity with the dismantled spine (compound 14) inhibits the gatekeeper mutation of BCR-ABL-T315I. These results demonstrate that mutation of the gatekeeper threonine is a common mechanism of activation for tyrosine kinases and provide structural insights to guide the development of next-generation inhibitors

Inhibition of the EGF receptor by binding of MIG6 to an activating kinase domain interface.

Members of the epidermal growth factor receptor family (EGFR/ERBB1, ERBB2/HER2, ERBB3/HER3 and ERBB4/HER4) are key targets for inhibition in cancer therapy. Critical for activation is the formation of an asymmetric dimer by the intracellular kinase domains, in which the carboxy-terminal lobe (C lobe) of one kinase domain induces an active conformation in the other. The cytoplasmic protein MIG6 (mitogen-induced gene 6; also known as ERRFI1) interacts with and inhibits the kinase domains of EGFR and ERBB2 (refs 3-5). Crystal structures of complexes between the EGFR kinase domain and a fragment of MIG6 show that a approximately 25-residue epitope (segment 1) from MIG6 binds to the distal surface of the C lobe of the kinase domain. Biochemical and cell-based analyses confirm that this interaction contributes to EGFR inhibition by blocking the formation of the activating dimer interface. A longer MIG6 peptide that is extended C terminal to segment 1 has increased potency as an inhibitor of the activated EGFR kinase domain, while retaining a critical dependence on segment 1. We show that signalling by EGFR molecules that contain constitutively active kinase domains still requires formation of the asymmetric dimer, underscoring the importance of dimer interface blockage in MIG6-mediated inhibition

Crystal Structure of the Cytoplasmic Domain of the Type I TGF β Receptor in Complex with FKBP12

AbstractActivation of the type I TGF β receptor (T β R-I) requires phosphorylation of a regulatory segment known as the GS region, located upstream of the serine/threonine kinase domain in the cytoplasmic portion of the receptor. The crystal structure of a fragment of unphosphorylated T β R-I, containing both the GS region and the catalytic domain, has been determined in complex with the FK506-binding protein FKBP12. T β R-I adopts an inactive conformation that is maintained by the unphosphorylated GS region. FKBP12 binds to the GS region of the receptor, capping the T β R-II phosphorylation sites and further stabilizing the inactive conformation of T β R-I. Certain structural features at the catalytic center of T β R-I are characteristic of tyrosine kinases rather than Ser/Thr kinases

Nucleotide-Induced Conformational Changes in an Isolated Escherichia coli DNA Polymerase III Clamp Loader Subunit

AbstractSliding clamps are loaded onto DNA by ATP-driven clamp loader complexes. The structure of the E. coli clamp loader in a nucleotide-free state has been determined previously. We now report crystal structures of a truncated form of the isolated γ-ATPase subunit, γ1–243, of the E. coli clamp loader, in nucleotide-free and bound forms. The γ subunit adopts a defined conformation when empty, in which the nucleotide binding site is blocked. The binding of either ATPγS or ADP, which are shown to bind with equal affinity to γ1–243, induces a change in the relative orientation of the two domains such that nucleotides can be accommodated. This change would break one of the γ:γ interfaces seen in the empty clamp loader complex, and may represent one step in the activation process

The structure of the leukemia drug imatinib bound to human quinone reductase 2 (NQO2)

<p>Abstract</p> <p>Background</p> <p>Imatinib represents the first in a class of drugs targeted against chronic myelogenous leukemia to enter the clinic, showing excellent efficacy and specificity for Abl, Kit, and PDGFR kinases. Recent screens carried out to find off-target proteins that bind to imatinib identified the oxidoreductase NQO2, a flavoprotein that is phosphorylated in a chronic myelogenous leukemia cell line.</p> <p>Results</p> <p>We examined the inhibition of NQO2 activity by the Abl kinase inhibitors imatinib, nilotinib, and dasatinib, and obtained IC₅₀values of 80 nM, 380 nM, and >100 μM, respectively. Using electronic absorption spectroscopy, we show that imatinib binding results in a perturbation of the protein environment around the flavin prosthetic group in NQO2. We have determined the crystal structure of the complex of imatinib with human NQO2 at 1.75 Å resolution, which reveals that imatinib binds in the enzyme active site, adjacent to the flavin isoalloxazine ring. We find that phosphorylation of NQO2 has little effect on enzyme activity and is therefore likely to regulate other aspects of NQO2 function.</p> <p>Conclusion</p> <p>The structure of the imatinib-NQO2 complex demonstrates that imatinib inhibits NQO2 activity by competing with substrate for the active site. The overall conformation of imatinib when bound to NQO2 resembles the folded conformation observed in some kinase complexes. Interactions made by imatinib with residues at the rim of the active site provide an explanation for the binding selectivity of NQO2 for imatinib, nilotinib, and dasatinib. These interactions also provide a rationale for the lack of inhibition of the related oxidoreductase NQO1 by these compounds. Taken together, these studies provide insight into the mechanism of NQO2 inhibition by imatinib, with potential implications for drug design and treatment of chronic myelogenous leukemia in patients.</p

Analysis of the role of PCNA-DNA contacts during clamp loading

<p>Abstract</p> <p>Background</p> <p>Sliding clamps, such as Proliferating Cell Nuclear Antigen (PCNA) in eukaryotes, are ring-shaped protein complexes that encircle DNA and enable highly processive DNA replication by serving as docking sites for DNA polymerases. In an ATP-dependent reaction, clamp loader complexes, such as the Replication Factor-C (RFC) complex in eukaryotes, open the clamp and load it around primer-template DNA.</p> <p>Results</p> <p>We built a model of RFC bound to PCNA and DNA based on existing crystal structures of clamp loaders. This model suggests that DNA would enter the clamp at an angle during clamp loading, thereby interacting with positively charged residues in the center of PCNA. We show that simultaneous mutation of Lys 20, Lys 77, Arg 80, and Arg 149, which interact with DNA in the RFC-PCNA-DNA model, compromises the ability of yeast PCNA to stimulate the DNA-dependent ATPase activity of RFC when the DNA is long enough to extend through the clamp. Fluorescence anisotropy binding experiments show that the inability of the mutant clamp proteins to stimulate RFC ATPase activity is likely caused by reduction in the affinity of the RFC-PCNA complex for DNA. We obtained several crystal forms of yeast PCNA-DNA complexes, measuring X-ray diffraction data to 3.0 Å resolution for one such complex. The resulting electron density maps show that DNA is bound in a tilted orientation relative to PCNA, but makes different contacts than those implicated in clamp loading. Because of apparent partial disorder in the DNA, we restricted refinement of the DNA to a rigid body model. This result contrasts with previous analysis of a bacterial clamp bound to DNA, where the DNA was well resolved.</p> <p>Conclusion</p> <p>Mutational analysis of PCNA suggests that positively charged residues in the center of the clamp create a binding surface that makes contact with DNA. Disruption of this positive surface, which had not previously been implicated in clamp loading function, reduces RFC ATPase activity in the presence of DNA, most likely by reducing the affinity of RFC and PCNA for DNA. The interaction of DNA is not, however, restricted to one orientation, as indicated by analysis of the PCNA-DNA co-crystals.</p

Crystal structure of a DNA polymerase sliding clamp from a Gram-positive bacterium

BACKGROUND: Sliding DNA clamps are processivity factors that are required for efficient DNA replication. DNA polymerases maintain proximity to nucleic acid templates by interacting with sliding clamps that encircle DNA and thereby link the polymerase enzyme to the DNA substrate. Although the structures of sliding clamps from Gram-negative bacteria (E. coli), eukaryotes, archaea, and T4-like bacteriophages are well-known, the structure of a sliding clamp from Gram-positive bacteria has not been reported previously. RESULTS: We have determined the crystal structure of the dimeric β subunit of the DNA polymerase III holoenzyme of Streptococcus pyogenes. The sliding clamp from this Gram-positive organism forms a ring-shaped dimeric assembly that is similar in overall structure to that of the sliding clamps from Gram-negative bacteria, bacteriophage T4, eukaryotes and archaea. The dimer has overall dimensions of ~90 Å × ~70 Å × ~25 Å with a central chamber that is large enough to accommodate duplex DNA. In comparison to the circular shape of other assemblies, the S. pyogenes clamp adopts a more elliptical structure. CONCLUSION: The sequences of sliding clamps from S. pyogenes and E. coli are only 23% identical, making the generation of structural models for the S. pyogenes clamp difficult in the absence of direct experimental information. Our structure of the S. pyogenes β subunit completes the catalog of clamp structures from all the major sequence grouping of sliding clamps. The more elliptical rather than circular structure of the S. pyogenes clamp implies that the topological nature of encircling DNA, rather than a precise geometric shape, is the most conserved aspect for this family of proteins

The crystal structure of the catalytic domain of a eukaryotic guanylate cyclase

<p>Abstract</p> <p>Background</p> <p>Soluble guanylate cyclases generate cyclic GMP when bound to nitric oxide, thereby linking nitric oxide levels to the control of processes such as vascular homeostasis and neurotransmission. The guanylate cyclase catalytic module, for which no structure has been determined at present, is a class III nucleotide cyclase domain that is also found in mammalian membrane-bound guanylate and adenylate cyclases.</p> <p>Results</p> <p>We have determined the crystal structure of the catalytic domain of a soluble guanylate cyclase from the green algae <it>Chlamydomonas reinhardtii </it>at 2.55 Å resolution, and show that it is a dimeric molecule.</p> <p>Conclusion</p> <p>Comparison of the structure of the guanylate cyclase domain with the known structures of adenylate cyclases confirms the close similarity in architecture between these two enzymes, as expected from their sequence similarity. The comparison also suggests that the crystallized guanylate cyclase is in an inactive conformation, and the structure provides indications as to how activation might occur. We demonstrate that the two active sites in the dimer exhibit positive cooperativity, with a Hill coefficient of ~1.5. Positive cooperativity has also been observed in the homodimeric mammalian membrane-bound guanylate cyclases. The structure described here provides a reliable model for functional analysis of mammalian guanylate cyclases, which are closely related in sequence.</p

A structural role for the PHP domain in E. coli DNA polymerase III.

RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are.BACKGROUND: In addition to the core catalytic machinery, bacterial replicative DNA polymerases contain a Polymerase and Histidinol Phosphatase (PHP) domain whose function is not entirely understood. The PHP domains of some bacterial replicases are active metal-dependent nucleases that may play a role in proofreading. In E. coli DNA polymerase III, however, the PHP domain has lost several metal-coordinating residues and is likely to be catalytically inactive. RESULTS: Genomic searches show that the loss of metal-coordinating residues in polymerase PHP domains is likely to have coevolved with the presence of a separate proofreading exonuclease that works with the polymerase. Although the E. coli Pol III PHP domain has lost metal-coordinating residues, the structure of the domain has been conserved to a remarkable degree when compared to that of metal-binding PHP domains. This is demonstrated by our ability to restore metal binding with only three point mutations, as confirmed by the metal-bound crystal structure of this mutant determined at 2.9 Å resolution. We also show that Pol III, a large multi-domain protein, unfolds cooperatively and that mutations in the degenerate metal-binding site of the PHP domain decrease the overall stability of Pol III and reduce its activity. CONCLUSIONS: While the presence of a PHP domain in replicative bacterial polymerases is strictly conserved, its ability to coordinate metals and to perform proofreading exonuclease activity is not, suggesting additional non-enzymatic roles for the domain. Our results show that the PHP domain is a major structural element in Pol III and its integrity modulates both the stability and activity of the polymerase