Peter K. Hwang

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.

Clathrin self-assembly is mediated by a tandemly repeated superhelix.

Clathrin is a triskelion-shaped cytoplasmic protein that polymerizes into a polyhedral lattice on intracellular membranes to form protein-coated membrane vesicles. Lattice formation induces the sorting of membrane proteins during endocytosis and organelle biogenesis by interacting with membrane-associated adaptor molecules. The clathrin triskelion is a trimer of heavy-chain subunits (1,675 residues), each binding a single light-chain subunit, in the hub domain (residues 1,074–1,675). Light chains negatively modulate polymerization so that intracellular clathrin assembly is adaptor-dependent. Here we report the atomic structure, to 2.6 Å resolution, of hub residues 1,210–1,516 involved in mediating spontaneous clathrin heavy-chain polymerization and light-chain association,. The hub fragment folds into an elongated coil of α-helices, and alignment analyses reveal a 145-residue motif that is repeated seven times along the filamentous leg and appears in other proteins involved in vacuolar protein sorting. The resulting model provides a three-dimensional framework for understanding clathrin heavy-chain self-assembly, light-chain binding and trimerization.

A protein phosphorylation switch at the conserved allosteric site in GP.

A phosphorylation-initiated mechanism of local protein refolding activates yeast glycogen phosphorylase (GP). Refolding of the phosphorylated amino-terminus was shown to create a hydrophobic cluster that wedges into the subunit interface of the enzyme to trigger activation. The phosphorylated threonine is buried in the allosteric site. The mechanism implicates glucose 6-phosphate, the allosteric inhibitor, in facilitating dephosphorylation by dislodging the buried covalent phosphate through binding competition. Thus, protein phosphorylation-dephosphorylation may also be controlled through regulation of the accessibility of the phosphorylation site to kinases and phosphatases. In mammalian glycogen phosphorylase, phosphorylation occurs at a distinct locus. The corresponding allosteric site binds a ligand activator, adenosine monophosphate, which triggers activation by a mechanism analogous to that of phosphorylation in the yeast enzyme.

Molecular analysis of GPH1, the gene encoding glycogen phosphorylase in Saccharomyces cerevisiae.

In yeast cells, the activity of glycogen phosphorylase is regulated by cyclic AMP-mediated phosphorylation of the enzyme. We have previously cloned the gene for glycogen phosphorylase (GPH1) in Saccharomyces cerevisiae. To assess the role of glycogen and phosphorylase-catalyzed glycogenolysis in the yeast life cycle, yeast strains lacking a functional GPH1 gene or containing multiple copies of the gene were constructed. GPH1 was found not to be an essential gene in yeast cells. Haploid cells disrupted in GPH1 lacked phosphorylase activity and attained higher levels of intracellular glycogen but otherwise were similar to wild-type cells. Diploid cells homozygous for the disruption were able to sporulate and give rise to viable ascospores. Absence of functional GPH1 did not impair cells from synthesizing and storing trehalose. Increases in phosphorylase activity of 10- to 40-fold were detected in cells carrying multiple copies of GPH1-containing 2 microns plasmid. Northern (RNA) analysis indicated that GPH1 transcription was induced at the late exponential growth phase, almost simultaneous with the onset of intracellular glycogen accumulation. Thus, the low level of glycogen in exponential cells was not primarily maintained through regulating the phosphorylation state of a constitutive amount of phosphorylase. GPH1 did not appear to be under formal glucose repression, since transcriptional induction occurred well in advance of glucose depletion from the medium.

Convergent and divergent evolution of regulatory sites in eukaryotic phosphorylases.

The activity of many proteins in eukaryotic cells is regulated by reversible covalent phosphorylation1. This regulatory modification is often linked to other allosteric controls within the same protein2, and such overlapping regulatory mechanisms are best characterized for glycogen phosphorylase (EC 2.4.1.1). Phosphorylases from different organisms or cell types exhibit markedly contrasting regulatory features3; this makes the enzyme attractive for studying the evolution of interacting molecular regulatory mechanisms4,5. Extensive biochemical and crystallographic studies of rabbit muscle phosphorylase have led to a characterization of five regulatory regions (phosphorylation, glycogen storage, AMP, glucose and purine sites)6–8. Here we report the complete primary structure of the yeast Saccharomyces cerevisiae glycogen phosphorylase, deduced from the sequence of the cloned gene. Regions that are highly conserved between muscle and yeast enzymes include the active site, the glycogen storage site and possibly the glucose and purine inhibition sites. Partial conservation of the residues involved in AMP-binding suggests a binding site for the yeast enzyme inhibitor, glucose 6-phosphate9,10. Other parts of the AMP site and the intersubunit contacts involved in AMP allostery are disrupted in the yeast enzyme by extreme sequence divergence. The poor alignment of amino termini and lack of homology at phosphorylation sites indicate that regulation by reversible phosphorylation evolved independently in yeast and vertebrate phosphorylases.

Actin Binding by Hip1 (Huntingtin-interacting Protein 1) and Hip1R (Hip1-related Protein) Is Regulated by Clathrin Light Chain

The huntingtin-interacting protein family members (Hip1 and Hip1R in mammals and Sla2p in yeast) link clathrin-mediated membrane traffic to actin cytoskeleton dynamics. Genetic data in yeast have implicated the light chain subunit of clathrin in regulating this link. To test this hypothesis, the biophysical properties of mammalian Hip1 and Hip1R and their interaction with clathrin light chain and actin were analyzed. The coiled-coil domains (clathrin light chain-binding) of Hip1 and Hip1R were found to be stable homodimers with no propensity to heterodimerize in vitro. Homodimers were also predominant in vivo, accounting for cellular segregation of Hip1 and Hip1R functions. Coiled-coil domains of Hip1 and Hip1R differed in their stability and flexibility, correlating with slightly different affinities for clathrin light chain and more markedly with effects of clathrin light chain binding on Hip protein-actin interactions. Clathrin light chain binding induced a compact conformation of both Hip1 and Hip1R and significantly reduced actin binding by their THATCH domains. Thus, clathrin is a negative regulator of Hip-actin interactions. These observations necessarily change models proposed for Hip protein function.

Complete cDNA sequence for rabbit muscle glycogen phosphorylase.

The cDNA for the nearly full‐length rabbit muscle glycogen phosphorylase mRNA has been isolated and sequenced. The cDNA is rich in G and C nucleotides. This feature is especially striking at the 3rd position of codons, where 86% of the 843 amino acid codons terminate with G or C. Methionine, presumably the initiation residue, is found at position—1, suggesting that the removal of only a single methionine residue precedes the amino‐terminal acetylation at serine. Eight differences between the deduced amino acid sequence and the previously determined protein sequence are discussed.

Conformation Switching of Clathrin Light Chain Regulates Clathrin Lattice Assembly

Clathrin-coated vesicle formation is responsible for membrane traffic to and from the endocytic pathway during receptor-mediated endocytosis and organelle biogenesis, influencing how cells relate to their environment. Generating these vesicles involves self-assembly of clathrin molecules into a latticed coat on membranes that recruits receptors and organizes protein machinery necessary for budding. Here we define a molecular mechanism regulating clathrin lattice formation by obtaining structural information from co-crystals of clathrin subunits. Low resolution X-ray diffraction data (7.9-9.0 A) was analyzed using a combination of molecular replacement with an energy-minimized model and noncrystallographic symmetry averaging. Resulting topological information revealed two conformations of the regulatory clathrin light chain bound to clathrin heavy chain. Based on protein domain positions, mutagenesis, and biochemical assays, we identify an electrostatic interaction between the clathrin subunits that allows the observed conformational variation in clathrin light chains to alter the conformation of the clathrin heavy chain and thereby regulates assembly.

Mechanism of Regulation in Yeast Glycogen Phosphorylase

The mechanism of yeast glycogen phosphorylase activation by covalent phosphorylation involves structural elements distinct from the mammalian homologs. To understand the role of the amino-terminal 39-residue extension in the phosphorylation control mechanism, mutants with 22 and 42 amino-terminal residues removed were expressed in Escherichia coli, and their properties were compared with the wild-type (WT) enzyme. The unphosphorylated WT enzyme had a specific activity of 0.1 unit/mg and was not activated significantly by the substrate, glucose 1-phosphate. Phosphorylation by protein kinase resulted in a 1300-fold activation. Glucose 6-phosphate inhibited the unphosphorylated enzyme more effectively than the phosphorylated form, and inhibition of the latter was cooperative. Glucose was a poor inhibitor for both the unphosphorylated and phosphorylated WT enzyme with K >300 mM. The rate of phosphorylation by protein kinase depended on substrates and interactions of the amino terminus. Maltoheptaose increased the rate of phosphorylation of the WT enzyme by yeast phosphorylase kinase 5-fold. The 22-residue deletion mutant (Nd22) had overall kinetic properties similar to the WT enzyme, except that Nd22 was a better substrate for the protein kinase and the rate of phosphorylation was unaffected by maltoheptaose. The 42-residue deletion mutant (Nd42), which lacks the phosphorylation site, was measurably active, although much less active than phosphorylated WT. Sedimentation equilibrium analysis indicated that the WT, Nd22, and Nd42 exist as tetramer, partially dissociated tetramer, and dimer, respectively. Phosphorylation of the WT and Nd22 converted both to dimer. The results indicated that the amino terminus affects quaternary structure and mediates activity regulation through conformational transition.

Clathrin self‐assembly involves coordinated weak interactions favorable for cellular regulation

The clathrin triskelion self‐assembles into a polyhedral coat surrounding membrane vesicles that sort receptor cargo to the endocytic pathway. A triskelion comprises three clathrin heavy chains joined at their C‐termini, extending into proximal and distal leg segments ending in a globular N‐terminal domain. In the clathrin coat, leg segments entwine into parallel and anti‐parallel interactions. Here we define the contributions of segmental interactions to the clathrin assembly reaction and measure the strength of their interactions. Proximal and distal leg segments were found to lack sufficient affinity to form stable homo‐ or heterodimers under assembly conditions. However, chimeric constructs of proximal or distal leg segments, trimerized by replacement of the clathrin trimerization domain with that of the invariant chain protein, were able to self‐assemble in reversible reactions. Thus clathrin assembly occurs because weak leg segment affinities are coordinated through trimerization, sharing a dependence on multiple weak interactions with other biopolymers. Such polymerization is sensitive to small environmental changes and is therefore compatible with cellular regulation of assembly, disassembly and curvature during formation of clathrin‐coated vesicles.