Julian J. Adams

Model for growth hormone receptor activation based on subunit rotation within a receptor dimer

Growth hormone is believed to activate the growth hormone receptor (GHR) by dimerizing two identical receptor subunits, leading to activation of JAK2 kinase associated with the cytoplasmic domain. However, we have reported previously that dimerization alone is insufficient to activate full-length GHR. By comparing the crystal structure of the liganded and unliganded human GHR extracellular domain, we show here that there is no substantial change in its conformation on ligand binding. However, the receptor can be activated by rotation without ligand by inserting a defined number of alanine residues within the transmembrane domain. Fluorescence resonance energy transfer (FRET), bioluminescence resonance energy transfer (BRET) and coimmunoprecipitation studies suggest that receptor subunits undergo specific transmembrane interactions independent of hormone binding. We propose an activation mechanism involving a relative rotation of subunits within a dimeric receptor as a result of asymmetric placement of the receptor-binding sites on the ligand.

Intrasteric control of AMPK via the γ1 subunit AMP allosteric regulatory site

AMP‐activated protein kinase (AMPK) is a αβγ heterotrimer that is activated in response to both hormones and intracellular metabolic stress signals. AMPK is regulated by phosphorylation on the α subunit and by AMP allosteric control previously thought to be mediated by both α and γ subunits. Here we present evidence that adjacent γ subunit pairs of CBS repeat sequences (after Cystathionine Beta Synthase) form an AMP binding site related to, but distinct from the classical AMP binding site in phosphorylase, that can also bind ATP. The AMP binding site of the γ1 CBS1/CBS2 pair, modeled on the structures of the CBS sequences present in the inosine monophosphate dehydrogenase crystal structure, contains three arginine residues 70, 152, and 171 and His151. The yeast γ homolog, snf4 contains a His151Gly substitution, and when this is introduced into γ1, AMP allosteric control is substantially lost and explains why the yeast snf1p/snf4p complex is insensitive to AMP. Arg70 in γ1 corresponds to the site of mutation in human γ2 and pig γ3 genes previously identified to cause an unusual cardiac phenotype and glycogen storage disease, respectively. Mutation of any of AMP binding site Arg residues to Gln substantially abolishes AMP allosteric control in expressed AMPK holoenzyme. The Arg/Gln mutations also suppress the previously described inhibitory properties of ATP and render the enzyme constitutively active. We propose that ATP acts as an intrasteric inhibitor by bridging the α and γ subunits and that AMP functions to derepress AMPK activity.

Nitrosylation of Human Glutathione Transferase P1-1 with Dinitrosyl Diglutathionyl Iron Complex in Vitro and in Vivo

We have recently shown that dinitrosyl diglutathionyl iron complex, a possible in vivo nitric oxide (NO) donor, binds with extraordinary affinity to one of the active sites of human glutathione transferase (GST) P1-1 and triggers negative cooperativity in the neighboring subunit of the dimer. This strong interaction has also been observed in the human Mu, Alpha, and Theta GST classes, suggesting a common mechanism by which GSTs may act as intracellular NO carriers or scavengers. We present here the crystal structure of GST P1-1 in complex with the dinitrosyl diglutathionyl iron ligand at high resolution. In this complex the active site Tyr-7 coordinates to the iron atom through its phenolate group by displacing one of the GSH ligands. The crucial importance of this catalytic residue in binding the nitric oxide donor is demonstrated by site-directed mutagenesis of this residue with His, Cys, or Phe residues. The relative binding affinity for the complex is strongly reduced in all three mutants by about 3 orders of magnitude with respect to the wild type. Electron paramagnetic resonance spectroscopy studies on intact Escherichia coli cells expressing the recombinant GST P1-1 enzyme indicate that bacterial cells, in response to NO treatment, are able to form the dinitrosyl diglutathionyl iron complex using intracellular iron and GSH. We hypothesize the complex is stabilized in vivo through binding to GST P1-1.

Calorimetric and structural studies of the nitric oxide carrier S-nitrosoglutathione bound to human glutathione transferase P1-1

The nitric oxide molecule (NO) is involved in many important physiological processes and seems to be stabilized by reduced thiol species, such as S‐nitrosoglutathione (GSNO). GSNO binds strongly to glutathione transferases, a major superfamily of detoxifying enzymes. We have determined the crystal structure of GSNO bound to dimeric human glutathione transferase P1‐1 (hGSTP1‐1) at 1.4 Å resolution. The GSNO ligand binds in the active site with the nitrosyl moiety involved in multiple interactions with the protein. Isothermal titration calorimetry and differential scanning calorimetry (DSC) have been used to characterize the interaction of GSNO with the enzyme. The binding of GSNO to wild‐type hGSTP1‐1 induces a negative cooperativity with a kinetic process concomitant to the binding process occurring at more physiological temperatures. GSNO inhibits wild‐type enzyme competitively at lower temperatures but covalently at higher temperatures, presumably by S‐nitrosylation of a sulfhydryl group. The C47S mutation removes the covalent modification potential of the enzyme by GSNO. These results are consistent with a model in which the flexible helix α2 of hGST P1‐1 must move sufficiently to allow chemical modification of Cys47. In contrast to wild‐type enzyme, the C47S mutation induces a positive cooperativity toward GSNO binding. The DSC results show that the thermal stability of the mutant is slightly higher than wild type, consistent with helix α2 forming new interactions with the other subunit. All these results suggest that Cys47 plays a key role in intersubunit cooperativity and that under certain pathological conditions S‐nitrosylation of Cys47 by GSNO is a likely physiological scenario.

Structure of Alzheimer's disease amyloid precursor protein copper-binding domain at atomic resolution

Amyloid precursor protein (APP) plays a central role in the pathogenesis of Alzheimers disease, as its cleavage generates the Abeta peptide that is toxic to cells. APP is able to bind Cu2+ and reduce it to Cu+ through its copper-binding domain (CuBD). The interaction between Cu2+ and APP leads to a decrease in Abeta production and to alleviation of the symptoms of the disease in mouse models. Structural studies of CuBD have been undertaken in order to better understand the mechanism behind the process. Here, the crystal structure of CuBD in the metal-free form determined to ultrahigh resolution (0.85 A) is reported. The structure shows that the copper-binding residues of CuBD are rather rigid but that Met170, which is thought to be the electron source for Cu2+ reduction, adopts two different side-chain conformations. These observations shed light on the copper-binding and redox mechanisms of CuBD. The structure of CuBD at atomic resolution provides an accurate framework for structure-based design of molecules that will deplete Abeta production.

Preventing serpin aggregation: The molecular mechanism of citrate action upon antitrypsin unfolding.

The aggregation of antitrypsin into polymers is one of the causes of neonatal hepatitis, cirrhosis, and emphysema. A similar reaction resulting in disease can occur in other human serpins, and collectively they are known as the serpinopathies. One possible therapeutic strategy involves inhibiting the conformational changes involved in antitrypsin aggregation. The citrate ion has previously been shown to prevent antitrypsin aggregation and maintain the protein in an active conformation; its mechanism of action, however, is unknown. Here we demonstrate that the citrate ion prevents the initial misfolding of the native state to a polymerogenic intermediate in a concentration‐dependent manner. Furthermore, we have solved the crystal structure of citrate bound to antitrypsin and show that a single citrate molecule binds in a pocket between the A and B β‐sheets, a region known to be important in maintaining antitrypsin stability.

Crystallization and preliminary crystallographic studies of the copper-binding domain of the amyloid precursor protein of Alzheimer's disease

Alzheimers disease is thought to be triggered by production of the amyloid beta (Abeta) peptide through proteolytic cleavage of the amyloid precursor protein (APP). The binding of Cu2+ to the copper-binding domain (CuBD) of APP reduces the production of Abeta in cell-culture and animal studies. It is expected that structural studies of the CuBD will lead to a better understanding of how copper binding causes Abeta depletion and will define a potential drug target. The crystallization of CuBD in two different forms suitable for structure determination is reported here.

Expression, purification, crystallization and preliminary X-ray diffraction analysis of chloride intracellular channel 2 (CLIC2)

The chloride intracellular channel (CLIC) family of proteins are unusual in that they can exist in either an integral membrane-channel form or a soluble form. Here, the expression, purification, crystallization and preliminary diffraction analysis of CLIC2, one of the least-studied members of this family, are reported. Human CLIC2 was crystallized in two different forms, both in the presence of reduced glutathione and both of which diffracted to better than 1.9 A resolution. Crystal form A displayed P2(1)2(1)2(1) symmetry, with unit-cell parameters a = 44.0, b = 74.7, c = 79.8 A. Crystal form B displayed P2(1) symmetry, with unit-cell parameters a = 36.0, b = 66.9, c = 44.1 A. Structure determination will shed more light on the structure and function of this enigmatic family of proteins.

Crystallization and preliminary X-ray diffraction analysis of the unliganded human growth hormone receptor.

The crystal structure of the extracellular domain of growth hormone receptor complexed to its ligand, growth hormone, has been known since 1992. However, no information exists for the unliganded form of the receptor. The human growth hormone receptors extracellular ligand-binding domain, encompassing amino-acid residues 1-238, has been expressed in Escherichia coli, purified by anion ion-exchange chromatography and crystallized in its unliganded state by the hanging-drop vapour-diffusion method in 100 mM HEPES pH 7.0 containing 27.5%(w/v) PEG 5000 monomethyl ether and 200 mM ammonium sulfate as the co-precipitants. The crystals belong to the othorhombic space group C222(1), have unit-cell parameters a = 99.7, b = 112.2, c = 93.2 A and diffract to 2.5 A resolution using synchrotron radiation. The crystal structure will shed light on the nature of any conformation changes that occur upon ligand binding and will provide information to develop potential low-molecular-weight agonists/antagonists to treat clinical diseases in which the growth hormone receptor is implicated.