CLIC1 (NCC27) is a member of the highly conserved class of chloride ion channels that exists in both soluble and integral membrane forms. Purified CLIC1 can integrate into synthetic lipid bilayers forming a chloride channel with similar properties to those observed in vivo. The structure of the soluble form of CLIC1 has been determined at 1.4-Å resolution. The protein is monomeric and structurally homologous to the glutathione S-transferase superfamily, and it has a redox-active site resembling glutaredoxin. The structure of the complex of CLIC1 with glutathione shows that glutathione occupies the redox-active site, which is adjacent to an open, elongated slot lined by basic residues. Integration of CLIC1 into the membrane is likely to require a major structural rearrangement, probably of the N-domain (residues 1-90), with the putative transmembrane helix arising from residues in the vicinity of the redox-active site. The structure indicates that CLIC1 is likely to be controlled by redox-dependent processes.Chloride ion channels, located both within the plasma membrane and other internal cell membranes (1, 2), are involved in diverse physiological processes. They are known to participate in the control of secretion and absorption of salt, regulation of membrane potentials, organellar acidification, and cell volume homeostasis (3). Malfunction in these channels can lead to severe disease states (4).Chloride channels fall into several classes based on their sequence relationships. The three best characterized classes are the ligand-gated receptor channels (␥-aminobutyric acid and glycine receptors), the cystic fibrosis transmembrane conductance regulator family, and the ClC chloride ion channels (1, 2). A new class of chloride ion channel, the "chloride intracellular channels" (CLICs), 1 has recently been characterized at a molecular level. To date, there are seven members of the CLIC family: CLIC1 (NCC27) (5), CLIC2 (6), CLIC3 (7), CLIC4 (8), CLIC5 (9), p64 (10), and parchorin (11). All of these proteins exist as soluble globular proteins that can form ion channels in organellar and plasma membranes (5,7,8,(12)(13)(14)(15). Five of the CLIC proteins are each composed of ϳ240 residues, while the longer p64 and parchorin consist of distinct amino-terminal domains followed by the 240-residue CLIC module. This module has recently been shown to share weak sequence homology with the glutathione S-transferase (GST) superfamily (16).The CLIC proteins are expressed in a wide variety of tissues and appear to have diverse physiological functions. p64 is associated with kidney function (17), while CLIC1 and CLIC4 appear to have a broad tissue distribution (5,8,18,19). Several CLICs interact with protein kinases (7,11,20). CLICs are associated with a variety of intracellular membranes including the nuclear membrane (5), the endoplasmic reticular membrane (8), large dense-core vesicles (19), mitochondria (21), trans-Golgi vesicles (22), and secretory vesicles (23). Parchorin forms the chloride channel in water-secreting cells,...

Cryptophytes are unicellular photosynthetic algae that use a lumenally located light-harvesting system, which is distinct from the phycobilisome structure found in cyanobacteria and red algae. One of the key components of this system is water-soluble phycoerythrin (PE) 545 whose expression is enhanced by low light levels. The crystal structure of the heterodimeric ␣ 1 ␣ 2 ␤␤ PE 545 from the marine cryptophyte Rhodomonas CS24 has been determined at 1.63-Å resolution. Although the ␤-chain structure is similar to the ␣ and ␤ chains of other known phycobiliproteins, the overall structure of PE 545 is novel with the ␣ chains forming a simple extended fold with an antiparallel ␤-ribbon followed by an ␣-helix. The two doubly linked ␤50͞␤61 chromophores (one on each ␤ subunit) are in van der Waals contact, suggesting that exciton-coupling mechanisms may alter their spectral properties. Each ␣ subunit carries a covalently linked 15,16-dihydrobiliverdin chromophore that is likely to be the final energy acceptor. The architecture of the heterodimer suggests that PE 545 may dock to an acceptor protein via a deep cleft and that energy may be transferred via this intermediary protein to the reaction center.Light-harvesting proteins increase the efficiency of photosynthetic organisms growing in low-light regimes. They act as antennae, capturing photons over a broad frequency spectrum and transferring energy to membrane-bound reaction centers (1). In cyanobacteria and red algae, the light-harvesting phycobiliproteins (PBPs) are water soluble and organized into phycobilisomes (large, multiprotein complexes bound to the stromal face of the thylakoids). Individual PBPs and phycobilisomes have been studied by x-ray crystallography (2-11) and electron microscopy (12, 13) as well as biochemically (14). The individual proteins are structurally conserved with a basic ␣␤ unit (referred to by convention as monomer) arranged around a 3-fold axis forming an (␣␤) 3 trimer. Cryptophyte algae also use PBPs to harvest light but these differ from those of the cyanobacteria and red algae in several significant ways (15). In any one species, there is only one type of PBP, either phycocyanin (PC) or phycoerythrin (PE); allophycocyanin is never present. The PC or PE is not organized into a phycobilisome but is instead located in the thylakoid lumen (16)(17)(18). Although the ␤ subunits of cryptophyte PBPs share a high degree of sequence identity with both the ␣ and ␤ subunits of cyanobacterial and red algal PBPs (19), the ␣ subunits are shorter, unrelated to other proteins in the sequence databases and carry a single, spectroscopically distinct bilin chromophore (20,21). Structurally, they also differ in being ␣ 1 ␣ 2 ␤␤ dimers rather than (␣␤) 3 trimers. We report here the structure at 1.63 Å of PE 545 from the marine cryptophyte Rhodomonas (formerly Chroomonas) CS24. METHODSProtein Preparation and Crystallization. PE 545 was purified (22) from Rhodomonas CS24 (Commonwealth Scientific and Industrial Research Organization, Division of Fishe...

Src tyrosine kinase has long been implicated in colon cancer but much remains to be learned about its substrates. The nuclear receptor hepatocyte nuclear factor 4α (HNF4α) has just recently been implicated in colon cancer but its role is poorly defined. Here we show that c-Src phosphorylates human HNF4α on three tyrosines in an interdependent and isoform-specific fashion. The initial phosphorylation site is a Tyr residue (Y14) present in the N-terminal A/B domain of P1-but not P2-driven HNF4α. Phospho-Y14 interacts with the Src SH2 domain, leading to the phosphorylation of two additional tyrosines in the ligand binding domain (LBD) in P1-HNF4α. Phosphomimetic mutants in the LBD decrease P1-HNF4α protein stability, nuclear localization and transactivation function. Immunohistochemical analysis of approximately 450 human colon cancer specimens (Stage III) reveals that P1-HNF4α is either lost or localized in the cytoplasm in approximately 80% of tumors, and that staining for active Src correlates with those events in a subset of samples. Finally, three SNPs in the human HNF4α protein, two of which are in the HNF4α F domain that interacts with the Src SH3 domain, increase phosphorylation by Src and decrease HNF4α protein stability and function, suggesting that individuals with those variants may be more susceptible to Src-mediated effects. This newly identified interaction between Src kinase and HNF4α has important implications for colon and other cancers.HNF4 isoforms | SH2 SH3 domain | SNP | Src kinase | tyrosine phosphorylation C olon cancer, the third most common malignancy in the United States, is a multifactorial disease that is influenced by both genetics and the environment (1, 2). c-Src is a nonreceptor tyrosine kinase that is strongly implicated in the development, growth, progression, and metastasis of several human cancers (3). In colon cancer, Src activation is associated with the early stages (4, 5) as well as progression and metastasis (6-8). Despite this long association with colon cancer, much remains to be learned about Src substrates (9).Hepatocyte nuclear factor 4alpha (HNF4α) (NR2A1) is a highly conserved member of the nuclear receptor superfamily with a recently identified endogenous ligand (linoleic acid) that binds in a reversible fashion (10, 11). HNF4α is best known for its role as a master regulator of liver-specific gene expression and as a key player in beta cells of the pancreas where it is mutated in an inherited form of type 2 diabetes (12)(13)(14). HNF4α is also expressed in kidney, stomach, and intestine; several recent papers also show an important role for HNF4α in the colon (15-20). There are two different promoters (P1 and P2) of HNF4A that are utilized in a temporal and tissue-specific fashion (11) (Fig. S1). While only P1-driven HNF4α (P1-HNF4α) is expressed in the adult liver, both P1-and P2-driven HNF4α (P2-HNF4α) are expressed in the adult intestine and colon (21, 22). Expression of P1-HNF4α is decreased in several human cancers including hepatocellular, gastric, renal, and...

A statistical analysis of interstrand interactions indicated that the shutter region can be used to discriminate between inhibitory and non-inhibitory serpins. This analysis implied that insertion of the RCL into betasheet A up to residue P8 is important for protease inhibition and hence the structure of the complex formed between the serpin and the target protease.