Robert J. Fletterick

The family of glycogen phosphorylases: structure and function

Glycogen phosphorylase plays a central role in the mobilization of carbohydrate reserves in a wide variety of organisms and tissues. While rabbit muscle phosphorylase remains the most studied and best characterized of phosphorylases, recombinant DNA techniques have led to the recent appearance of primary sequence data for a wide variety of phosphorylase enzymes. The functional properties of rabbit muscle phosphorylases are reviewed and then compared to properties of phosphorylases from other tissues and organisms. Tissue expression patterns and the chromosomal localization of mammalian phosphorylases are described. Differences in functional properties among phosphorylases are related to new structural information. Evolutionary relationships among phosphorylases as afforded by comparative analysis of proteins and gene sequences are discussed.

Detection of a trypsin-like serine protease domain in flaviviruses and pestviruses

We propose through a sequence and structural-pattern analysis that a protein domain of undefined function encoded by the enveloped RNA flavi- and pestivruses is a Ser active-center enzyme related to the cellular trypsin family. A further homology is emphasized with the group of (Cys active-center) viral proteases encoded by nonenveloped RNA viruses of the picorna-, como-, nepo-, and potyvirus classes. Structural modeling of the putative flaviviral protease domain suggests amino acids that are crucial for catalytic activity and substrate binding.

Recognition and Accommodation at the Androgen Receptor Coactivator Binding Interface

Prostate cancer is a leading killer of men in the industrialized world. Underlying this disease is the aberrant action of the androgen receptor (AR). AR is distinguished from other nuclear receptors in that after hormone binding, it preferentially responds to a specialized set of coactivators bearing aromatic-rich motifs, while responding poorly to coactivators bearing the leucine-rich “NR box” motifs favored by other nuclear receptors. Under normal conditions, interactions with these AR-specific coactivators through aromatic-rich motifs underlie targeted gene transcription. However, during prostate cancer, abnormal association with such coactivators, as well as with coactivators containing canonical leucine-rich motifs, promotes disease progression. To understand the paradox of this unusual selectivity, we have derived a complete set of peptide motifs that interact with AR using phage display. Binding affinities were measured for a selected set of these peptides and their interactions with AR determined by X-ray crystallography. Structures of AR in complex with FxxLF, LxxLL, FxxLW, WxxLF, WxxVW, FxxFF, and FxxYF motifs reveal a changing surface of the AR coactivator binding interface that permits accommodation of both AR-specific aromatic-rich motifs and canonical leucine-rich motifs. Induced fit provides perfect mating of the motifs representing the known family of AR coactivators and suggests a framework for the design of AR coactivator antagonists.

A Model for the Microtubule-Ncd Motor Protein Complex Obtained by Cryo-Electron Microscopy and Image Analysis

Kinesin motors convert chemical energy from ATP hydrolysis into unidirectional movement. To understand how kinesin motors bind to and move along microtubules, we fit the atomic structure of the motor domain of Ncd (a kinesin motor involved in meiosis and mitosis) into three-dimensional density maps of Ncd-microtubule complexes calculated by cryo-electron microscopy and image analysis. The model reveals that Ncd shares an extensive interaction surface with the microtubule, and that a portion of the binding site involves loops that contain conserved residues. In the Ncd dimer, the microtubule-bound motor domain makes intimate contact with its partner head, which is dissociated from the microtubule. This head-head interaction may be important in positioning the dissociated head to take a step to the next binding site on the microtubule protofilament.

Structural Basis for Ligand-Independent Activation of the Orphan Nuclear Receptor LRH-1

The orphan nuclear receptors SF-1 and LRH-1 are constitutively active, but it remains uncertain whether their activation is hormone dependent. We report the crystal structure of the LRH-1 ligand binding domain to 2.4 A resolution and find the receptor to be a monomer that adopts an active conformation with a large but empty hydrophobic pocket. Adding bulky side chains into this pocket resulted in full or greater activity suggesting that, while LRH-1 could accommodate potential ligands, these are dispensable for basal activity. Constitutive LRH-1 activity appears to be conferred by a distinct structural element consisting of an extended helix 2 that provides an additional layer to the canonical LBD fold. Mutating the conserved arginine in helix 2 reduced LRH-1 receptor activity and coregulator recruitment, consistent with the partial loss-of-function phenotype exhibited by an analogous SF-1 human mutant. These findings illustrate an alternative structural strategy for nuclear receptor stabilization in the absence of ligand binding.

The case for a common ancestor: kinesin and myosin motor proteins and G proteins.

Recent studies have shown surprising structural and functional similarities between the motor domains of kinesin and myosin. Common features have also been described for motor proteins and G proteins. Despite these similarities, the evolutionary relationships between these proteins, even among the motor proteins, has not been obvious, since the topological connectivities of the core overlapping structural elements in these transducing proteins are not identical to one another. Using secondary structure topology, comparison of functional domains and active site chemistry as criteria for relatedness, we propose a set of rules for determining potential evolutionary relationships between proteins showing little or no sequence identity. These rules were used to explore the evolutionary relationship between kinesin and myosin, as well as between motor proteins and other phosphate-loop (P-loop) containing nucleotide- binding proteins. We demonstrate that kinesin and myosin show significant chemical conservations within and outside of the active site, and present an evolutionary scheme that produces their respective topologies from a hypothetical ancestral protein. We also show that, when compared with various other P-loop-containing proteins, the cytoskeletal motors are most similar to G proteins with respect to topology and active site chemistry. We conclude that kinesin and myosin, and possibly G proteins, are probably directly related via divergent evolution from a common core nucleotide-binding motif, and describe the likely topology of this ancestor. These proteins use similar chemical and physical mechanisms to both sense the state of the nucleotide bound in the active site, and then transmit these changes to protein partners. The different topologies can be accounted for by unique genetic insertions that add to the edge of a progenitor protein structure and do not disrupt the hydrophobic core.

A role for helix 3 of the TRβ ligand-binding domain in coactivator recruitment identified by characterization of a third cluster of mutations in resistance to thyroid hormone

Resistance to thyroid hormone (RTH) has hitherto been associated with thyroid hormone β receptor (TRβ) mutations which cluster in two regions (αα 310–353 and αα 429–461) of the hormone‐binding domain and closely approximate the ligand‐binding cavity. Here, we describe a third cluster of RTH mutations extending from αα 234–282 which constitute a third boundary of the ligand pocket. One mutant, T277A, exhibits impaired transactivation which is disproportionate to its mildly reduced ligand affinity (Ka). T3‐dependent recruitment of coactivators (SRC‐1, ACTR) by mutant receptor–RXR heterodimers was reduced in comparison with wild‐type. Cotransfection of SRC‐1 restored transactivation by T277A. In the TRβ crystal structure this helix 3 residue is surface‐exposed and is in close proximity to residues L454 and E457 in helix 12 which are known to be critical for coactivator interaction, suggesting that they all constitute part of a receptor–coactivator interface. The transcriptional function of other mutants (A234T, R243W/Q, A268D, Δ276I, A279V, R282S) in this cluster correlated with their reduced Ka and they inhibited wild‐type TRβ action in a dominant negative manner. DNA binding, heterodimerization and corepressor recruitment were preserved in all mutants, signifying the importance of these attributes for dominant negative activity and correlating with the absence of natural mutations in regions bordering the third cluster which mediate these functions.

Structure of maltoheptaose by difference Fourier methods and a model for glycogen

Abstract The structure of the glucose heptamer, maltoheptaose, has been determined by difference Fourier analysis at 0.25 nm resolution through its binding to phosphorylase a . It is a left-handed helical structure, with 6.5 glucose residues per turn and a rise per residue of 2.4 A. The molecule shows short-range order when no protein is present to stabilize its conformation in solution. With one exception, the individual torsion angles between sugar residues vary over a narrow range and preserve a good O (2) O (3′) hydrogen bond. The length of an individual chain for glycogen can be extrapolated from the maltoheptaose data and agrees well with the size for glycogen predicted by the Whelan model.

Phosphorylation and Intramolecular Stabilization of the Ligand Binding Domain in the Nuclear Receptor Steroidogenic Factor 1

ABSTRACT Steroidogenic factor 1 (SF-1) is an orphan nuclear receptor with no known ligand. We showed previously that phosphorylation at serine 203 located N′-terminal to the ligand binding domain (LBD) enhanced cofactor recruitment, analogous to the ligand-mediated recruitment in ligand-dependent receptors. In this study, results of biochemical analyses and an LBD helix assembly assay suggest that the SF-1 LBD adopts an active conformation, with helices 1 and 12 packed against the predicted alpha-helical bundle, in the apparent absence of ligand. Fine mapping of the previously defined proximal activation function in SF-1 showed that the activation function mapped fully to helix 1 of the LBD. Limited proteolyses demonstrate that phosphorylation of S203 in the hinge region mimics the stabilizing effects of ligand on the LBD. Moreover, similar effects were observed in an SF-1/thyroid hormone LBD chimera receptor, illustrating that the S203 phosphorylation effects are transferable to a heterologous ligand-dependent receptor. Our collective data suggest that the hinge together with helix 1 is an individualized specific motif, which is tightly associated with its cognate LBD. For SF-1, we find that this intramolecular association and hence receptor activity are further enhanced by mitogen-activated protein kinase phosphorylation, thus mimicking many of the ligand-induced changes observed for ligand-dependent receptors.

Shape and Specificity in Mammalian 15-Lipoxygenase Active Site THE FUNCTIONAL INTERPLAY OF SEQUENCE DETERMINANTS FOR THE REACTION SPECIFICITY

Previous mutagenesis studies along with molecular modeling using the x-ray coordinates of the rabbit 15-lipoxygenase have led to the suggestion that the size of the substrate binding pocket may play an essential role in determining the oxygenation specificity of 5-, 12-, and 15-lipoxygenases. Based on the x-ray crystal structure of rabbit 15-lipoxygenase, Ile593 appeared to be important in defining size and shape of the substrate-binding site in 15-lipoxygenases. We found that substitution of Ile593 with alanine shifted the positional specificity of this enzyme toward 12-lipoxygenation. To compare the importance of position 593 with previously defined determinants for the oxygenation specificity, we introduced small (alanine-scan) or large amino acids (phenylalanine-scan) at critical positions surrounding the putative fatty acid-binding site, so that the volume of the pocket was either increased or decreased. Enlargement or alteration in packing density within the substrate binding pocket in the rabbit 15-lipoxygenase increased the share of 12-lipoxygenase products, whereas a smaller active site favored 15-lipoxygenation. Simultaneous substitution of both large and small residues in the context of either a 15- or 12-lipoxygenase indicated that there is a functional interplay of the sequence determinants for lipoxygenation specificity. If the 15-lipoxygenase active site is enlarged excessively, however, no lipoxygenation was observed anymore. Together these results indicate the importance of the overall size and shape of the arachidonic acid binding pocket in defining the specificity of lipoxygenase reaction.