Mark A. Fleming

X-RAY STRUCTURE OF CALCINEURIN INHIBITED BY THE IMMUNOPHILIN-IMMUNOSUPPRESSANT FKBP12-FK506 COMPLEX

The X-ray structure of the ternary complex of a calcineurin A fragment, calcineurin B, FKBP12, and the immunosuppressant drug FK506 (also known as tacrolimus) has been determined at 2.5 A resolution, providing a description of how FK506 functions at the atomic level. In the structure, the FKBP12-FK506 binary complex does not contact the phosphatase active site on calcineurin A that is more than 10 A removed. Instead, FKBP12-FK506 is so positioned that it can inhibit the dephosphorylation of its macromolecular substrates by physically hindering their approach to the active site. The ternary complex described here represents the three-dimensional structure of a Ser/Thr protein phosphatase and provides a structural basis for understanding calcineurin inhibition by FKBP12-FK506.

Crystal structure of JNK3: a kinase implicated in neuronal apoptosis.

BACKGROUND The c-Jun N-terminal kinases (JNKs) are members of the mitogen-activated protein (MAP) kinase family, and regulate signal transduction in response to environmental stress. Activation and nuclear localization of JNK3, a neuronal-specific isoform of JNK, has been associated with hypoxic and ischemic damage of CA1 neurons in the hippocampus. Knockout mice lacking JNK3 showed reduced apoptosis of hippocampal neurons and reduced seizure induced by kainic acid, a glutamate-receptor agonist. Thus, JNK3 may be important in the pathology of neurological disorders and is of significant medical interest. RESULTS We report here the structure of unphosphorylated JNK3 in complex with adenylyl imidodiphosphate, an ATP analog. JNK3 has a typical kinase fold, with the ATP-binding site situated within a cleft between the N- and C-terminal domains. In contrast to other known MAP kinase structures, the ATP-binding site of JNK3 is well ordered; the glycine-rich nucleotide-binding sequence forms a beta-strand-turn-beta-strand structure over the nucleotide. Unphosphorylated JNK3 assumes an open conformation, in which the N- and C-terminal domains are twisted apart relative to their positions in cAMP-dependent protein kinase. The rotation leads to the misalignment of some of the catalytic residues. The phosphorylation lip of JNK3 partially blocks the substrate-binding site. CONCLUSIONS This is the first JNK structure to be determined, providing a unique opportunity to compare structures from the three MAP kinase subfamilies. The structure reveals atomic-level details of the shape of JNK3 and the interactions between the kinase and the nucleotide. The misalignment of catalytic residues and occlusion of the active site by the phosphorylation lip may account for the low activity of unphosphorylated JNK3. The structure provides a framework for understanding the substrate specificity of different JNK isoforms, and should aid the design of selective JNK3 inhibitors.

Conformational Changes and Stabilization of Inosine 5′-Monophosphate Dehydrogenase Associated with Ligand Binding and Inhibition by Mycophenolic Acid

The effects of substrate, product, and inhibitor (mycophenolic acid) binding on the conformation and stability of hamster type II inosine 5′-monophosphate dehydrogenase (IMPDH) have been examined. The protein in various states of ligand occupancy was compared by analyzing susceptibility to in vitro proteolysis, the degree of binding of a hydrophobic fluorescent dye, secondary structure content as determined by far-UV circular dichroism spectra, and urea-induced denaturation curves. These analysis methods revealed consistent evidence that IMPDH undergoes a local reorganization when IMP or XMP bind. NAD+ produced no such effect. In fact, no evidence was found for NAD+ binding independently of IMP. It is proposed that IMPDH adopts an open conformation around its nucleotide binding sites in the absence of substrates and that binding of IMP stabilizes a closed conformation that has a higher affinity for NAD+. The data also suggest the enzyme remains in the closed configuration throughout the catalytic steps and then reverts to the open conformation with XMP release, thereby consummating the enzyme cycle. Mycophenolic acid inhibition appeared to impart even greater stability. We propose that localized conformational changes occur during the normal and mycophenolic acid-inhibited reaction sequences of IMPDH.

Purification and characterization of the NS3 serine protease domain of hepatitis C virus expressed in Saccharomyces cerevisiae.

cDNA encoding the putative core of the hepatitis C virus NS3 serine protease domain (residues 1-181 of NS3; NS3 (181)) was expressed as an N-terminally (His)6-tagged fusion protein in Saccharomyces cerevisiae. NS3 (181) protease activity was found in soluble cell lysates, and the N-terminal metal-chelating domain facilitated the efficient purification of active enzyme, using immobilized metal affinity chromatography. The purified NS3(181), protease activity was characterized by assaying the trans-cleavage of in vitro transcription-translation generated substrates, and subsequently a previously unobserved cleavage site within the NS5A region was identified. The inhibitory effect of known protease inhibitors was also examined. It is hoped that availability of this method for the expression and purification of the NS3(181) protease will facilitate the development of anti-hepatitis C therapies.

Kinetic mechanism and ATP‐binding site reactivity of p38γ MAP kinase

Activated p38γ MAP kinase exhibited significant basal ATPase activity in the absence of a kinase substrate, and addition of a phosphoacceptor substrate increased k cat/K m>20‐fold. AMP‐PCP was competitive with ATP binding and non‐competitive with phosphoacceptor substrate binding. The nucleotide binding site affinity label 5′‐(p‐fluorosulfonylbenzoyl)adenosine (FSBA) bound stoichiometrically at Lys‐56 in the ATP site of both unphosphorylated and activated p38γ. AMP‐PCP only protected the activated enzyme from FSBA inactivation, implying that AMP‐PCP does not bind unphosphorylated p38γ. Basal ATPase activities were also observed for activated p38α, ERK2 and JNK3 suggesting that the enzymatic mechanism may be similar for all classes of MAP kinases.

A signature for immune response correlates with HCV treatment outcome in Caucasian subjects

This data article contains Supplementary material for a published research article describing a whole-blood proteomic signature that predicts treatment outcome for subjects infected with hepatitis C virus (HCV) [1]. The proteomic signature is derived from whole-blood samples from subjects infected with HCV. The article includes detailed experimental and computational methods used in the analysis. The article also includes tables of demographic and other information about the subjects. Finally, the article includes several figures and tables showing detailed results of the analyses (e.g. lists of identified proteins and coefficients/ROC curves for the regression models).