Focco van den Akker

Structure of the dimerized hormone-binding domain of a guanylyl-cyclase-coupled receptor.

The atrial natriuretic peptide (ANP) hormone is secreted by the heart in response to an increase in blood pressure. ANP exhibits several potent anti-hypertensive actions in the kidney, adrenal gland and vascular system. These actions are induced by hormone binding extracellularly to the ANP receptor, thereby activating its intracellular guanylyl cyclase domain for the production of cyclic GMP. Here we present the crystal structure of the glycosylated dimerized hormone-binding domain of the ANP receptor at 2.0-Å resolution. The monomer comprises two interconnected subdomains, each encompassing a central β-sheet flanked by α-helices, and exhibits the type I periplasmic binding protein fold. Dimerization is mediated by the juxtaposition of four parallel helices, arranged two by two, which brings the two protruding carboxy termini into close relative proximity. From affinity labelling and mutagenesis studies, the ANP-binding site maps to the side of the dimer crevice and extends to near the dimer interface. A conserved chloride-binding site is located in the membrane distal domain, and we found that hormone binding is chloride dependent. These studies suggest mechanisms for hormone activation and the allostery of the ANP receptor.

Association of STATs with relatives and friends.

Members of the STAT family of transcription factors are present in species as diverse as mammals, insects and slime molds. Discovered as mediators of interferon-induced signals, the STATs were later shown to drive many different ligand-induced responses through receptor-induced tyrosine phosphorylation and dimerization. STAT1 also functions as a transcription factor, essential for the efficient constitutive expression of certain genes, without needing tyrosine phosphorylation, and phosphorylated STAT1 dimers mediate suppression - rather than activation - of some genes. STATs are present in the cytoplasm of untreated cells in multiprotein complexes, which might aid in their nuclear translocation and differential binding to DNA, thus contributing to the specificity of STAT action. This review explores the diverse protein-protein interactions that underlie the multiple functions of the STATs.

AIPL1, a protein implicated in Leber's congenital amaurosis, interacts with and aids in processing of farnesylated proteins

The most common form of blindness at birth, Lebers congenital amaurosis (LCA), is inherited in an autosomal recessive fashion. Mutations in six different retina-specific genes, including a recently discovered gene, AIPL1, have been linked to LCA in humans. To understand the molecular basis of LCA caused by aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1) mutations, and to elucidate the normal function of AIPL1, we performed a yeast two-hybrid screen using AIPL1 as bait. The screen demonstrated that AIPL1 interacts specifically with farnesylated proteins. Mutations in AIPL1 linked to LCA compromise this activity. These findings suggest that the essential function of AIPL1 within photoreceptors requires interactions with farnesylated proteins. Analysis of isoprenylation in cultured human cells shows that AIPL1 enhances the processing of farnesylated proteins. Based on these findings, we propose that AIPL1 interacts with farnesylated proteins and plays an essential role in processing of farnesylated proteins in retina.

A reinterpretation of the dimerization interface of the N-terminal Domains of STATs

The crystal structures of the N‐terminal domain (N‐domain) and the core region of the STAT family of transcription factors have been determined previously. STATs can form cooperative higher order structures (tetramers or higher oligomers) while bound to DNA. The crystal packing in the STAT4 N‐domain crystal structure, determined at 1.5 Å resolution, suggests two alternate organizations of the N‐domain dimer. We now present the results of site directed mutagenesis of residues predicted to be involved at each dimer interface. Our results indicate that the dimer interface suggested earlier as being physiologically relevant is, in fact, unlikely to be so. Given the alternative model for the N‐domain dimer, the ability of the N‐domain to mediate interactions of two STAT dimers on DNA remains unchanged.

Structural insights into the ligand binding domains of membrane bound guanylyl cyclases and natriuretic peptide receptors

Membrane bound guanylyl cyclases are single chain transmembrane receptors that produce the second messenger cGMP by either intra- or extracellular stimuli. This class of type I receptors contain an intracellular catalytic guanylyl cyclase domain, an adjacent kinase-like domain and an extracellular ligand binding domain though some receptors have their ligands yet to be identified. The most studied member is the atrial natriuretic peptide (ANP) receptor, which is involved in blood pressure regulation. Extracellular ANP binding induces a conformational change thereby activating the pre-oligomerized receptor leading to the production of cGMP. The recent crystal structure of the dimerized hormone binding domain of the ANP receptor provides a first three-dimensional view of this domain and can serve as a basis to structurally analyze mutagenesis, cross-linking, and genetic studies of this class of receptors as well as a non-catalytic homolog, the clearance receptor. The fold of the ligand binding domain is that of a bilobal periplasmic binding protein (PBP) very similar to that of the Leu/Ile/Val binding protein, AmiC, multi-domain transmembrane metabotropic glutamate receptors, and several DNA binding proteins such as the lactose repressor. Unlike these structural homologs, the guanylyl cyclase receptors bind much larger molecules at a site seemingly remote from the usual small molecule binding site in periplasmic binding protein folds. Detailed comparisons with these structural homologs offer insights into mechanisms of signal transduction and allosteric regulation, and into the remarkable usage of the periplasmic binding protein fold in multi-domain receptors/proteins.

Crystal structure of a new heat-labile enterotoxin, LT-IIb.

BACKGROUND Cholera toxin from Vibrio cholerae and the type I heat-labile enterotoxins (LT-Is) from Escherichia coli are oligomeric proteins with AB5 structures. The type II heat-labile enterotoxins (LT-IIs) from E. coli are structurally similar to, but antigenically distinct from, the type I enterotoxins. The A subunits of type I and type II enterotoxins are homologous and activate adenylate cyclase by ADP-ribosylation of a G protein subunit, G8 alpha. However, the B subunits of type I and type II enterotoxins differ dramatically in amino acid sequence and ganglioside-binding specificity. The structure of LT-IIb was determined both as a prototype for other LT-IIs and to provide additional insights into structure/function relationships among members of the heat-labile enterotoxin family and the superfamily of ADP-ribosylating protein toxins. RESULTS The 2.25 A crystal structure of the LT-IIb holotoxin has been determined. The structure reveals striking similarities with LT-I in both the catalytic A subunit and the ganglioside-binding B subunits. The latter form a pentamer which has a central pore with a diameter of 10-18 A. Despite their similarities, the relative orientation between the A polypeptide and the B pentamer differs by 24 degrees in LT-I and LT-IIb. A common hydrophobic ring was observed at the A-B5 interface which may be important in the cholera toxin family for assembly of the AB5 heterohexamer. A cluster of arginine residues at the surface of the A subunit of LT-I and cholera toxin, possibly involved in assembly, is also present in LT-IIb. The ganglioside receptor binding sites are localized, as suggested by mutagenesis, and are in a position roughly similar to the sites where LT-I binds its receptor. CONCLUSIONS The structure of LT-IIb provides insight into the sequence diversity and structural similarity of the AB5 toxin family. New knowledge has been gained regarding the assembly of AB5 toxins and their active-site architecture.

Adenovirus E1A Down-regulates LMP2 Transcription by Interfering with the Binding of Stat1 to IRF1

The LMP2 gene, which encodes a protein required for efficient presentation of viral antigens, requires both unphosphorylated Stat1 and IRF1 for basal expression. LMP2 expression is down-regulated by the adenovirus protein E1A, which binds to Stat1 and CBP/p300, and by the mutant E1A protein RG2, which binds to Stat1 but not to CBP/p300, but not by the mutant protein Δ2–36, which does not bind to either Stat1 or CBP/p300. Stat1 and IRF1 associate in untreated cells and bind as a complex to the overlapping ICS-2/GAS element of the LMP2 promoter. E1A interferes with the formation of this complex by occupying domains of Stat1 that bind to IRF1. These results reveal how adenovirus infection attenuates LMP2 expression, thereby interfering with the presentation of viral antigens.

Protein engineering studies of A‐chain loop 47‐56 of Escherichia coli heat‐labile enterotoxin point to a prominent role of this loop for cytotoxicity

Heat‐labile enterotoxin (LT), produced by enterotoxigenic Escherichia coli, is a close relative of cholera toxin (CT). These two toxins share approximately 80% sequence identity, and consists of one 240‐residue A chain and five 103‐residue B subunits. The B pentamer is responsible for GM1 receptor recognition, whereas the A subunit carries out an ADP‐ribosylation of an arginine residue in the G protein, GSα, in the epithelial target cell. This paper explores the importance of specific amino acids in loop 47–56 of the A subunit. This loop was observed to be highly mobile in the inactive R7K mutant of the A subunit. The position of the loop in wild‐type protein is such that it might require considerable reorganization during substrate binding and is likely to have a crucial role in substrate binding. Five single‐site substitutions have been made in the LT‐A subunit 47–56 loop to investigate its possible role in the enzymatic activity and toxicity of LT and CT. The wild‐type residues Thr‐50 and Val‐53 were replaced either by a glycine or by a proline. The glycine substitutions were intended to increase the mobility of this active‐site loop, and the proline substitutions were intended to decrease the mobility of this same loop by restricting the accessible conformational space. Under the hypothesis that mobility of the loop is important for catalysis, the glycine‐substitution mutants T50G and V53G would be expected to exhibit activity equal to or greater than that of the wild‐type A subunit, while the proline substitution mutants T50P and T53P would be less active. Cytotoxicity assays showed, however, that all four of these mutants were considerably less active than wild‐type LT. These results lend support for assignment of a prominent role to loop 47–56 in catalysis by LT and CT.

Expression and crystallization of several forms of the Propionibacterium shermanii transcarboxylase 5S subunit.

The dimeric outer 5S subunit of transcarboxylase has been expressed in three different forms and crystallized: native 5S, 5S-His(6) and selenomethione-5S-His(6). All the crystals have an orthorhombic space group, but while native 5S forms primitive orthorhombic crystals, 5S-His(6) crystals are either C-centered or primitive and SeMet-5S-His(6) crystals are C-centered. Crystallization of native 5S requires the addition of lithium sulfate, whereas this salt prevented crystallization of 5S-His(6). All 5S crystals diffract to approximately 2.0 A resolution with synchrotron radiation. Efforts are under way to solve the structure of SeMet-5S-His(6) using MAD.

E. Coli Heat Labile Enterotoxin and Cholera Toxin B-Pentamer—Crystallographic Studies of Biological Activity

The heat-labile enterotoxins from E. coli are part of a larger class of bacterial toxins whose members are collectively responsible for a variety of diseases known since ancient times and still afflicting the world today. Most notable of these is cholera, but also among them are dysentery, whooping cough, traveler’s diarrhea, and the more recently characterized hemolytic uremic syndrome (“hamburger disease”). Although the specific cellular targets, enzymatic activity, and biological mode of action of these toxins vary, their evolutionary kinship may be traced both through sequence homology and a shared protein quaternary structure. Members of this class of toxins are AB5 hexameric assemblies. Each toxin’s catalytic activity is carried on the A subunit, while the pentamer of B subunits mediates receptor recognition and cell surface binding. Atomic resolution structures have been determined for several of the AB5 toxins. Their structural and functional relationships have been reviewed recently by Burnette1 and Merritt and Hol.2