RS1, a discoidin domain-containing retinal cell adhesion protein associated with X-linked retinoschisis, exists as a novel disulfide-linked octamer

RS1, also known as retinoschisin, is an extracellular protein that plays a crucial role in the cellular organization of the retina. Mutations in RS1 are responsible for X-linked retinoschisis, a common, early-onset macular degeneration in males that results in a splitting of the inner layers of the retina and severe loss in vision. RS1 is assembled and secreted from photoreceptors and bipolar cells as a homo-oligomeric protein complex. Each subunit consists of a 157-amino acid discoidin domain flanked by two small segments of 39 and 5 amino acids. To begin to understand how the structure of RS1 relates to its role in retinal cell adhesion and X-linked retinoschisis, we have determined the subunit organization and disulfide bonding pattern of RS1 by SDS gel electrophoresis, velocity sedimentation, and mass spectrometry. Our results indicate that RS1 exists as a novel octamer in which the eight subunits are joined together by Cys(59)-Cys(223) intermolecular disulfide bonds. Subunits within the octamer are further organized into dimers mediated by Cys(40)-Cys(40) bonds. These cysteines lie just outside the discoidin domain indicating that these flanking segments primarily function in the octamerization of RS1. Within the discoidin domain, two cysteine pairs (Cys(63)-Cys(219) and Cys(110)-Cys(142)) form intramolecular disulfide bonds that are important in protein folding, and one cysteine (Cys(83)) exists in its reduced state. Because mutations that disrupt subunit assembly cause X-linked retinoschisis, the assembly of RS1 into a disulfide-linked homo-octamer appears to be critical for its function as a retinal cell adhesion protein.     

Defective discoidin domain structure,subunit assembly,and endoplasmic reticulum processing of retinoschisin are primary mechanisms responsible for X-linked retinoschisis

Retinoschisin is a 24-kDa discoidin domain-containing protein that is secreted from photoreceptor and bipolar cells as a large disulfide-linked multisubunit complex. It functions as a cell adhesion protein to maintain the cellular organization and synaptic structure of the retina. Over 125 different mutations in the RS1 gene are associated with X-linked juvenile retinoschisis, the most common form of early onset macular degeneration in males. To identify molecular determinants important for retinoschisin structure and function and elucidate molecular and cellular mechanisms responsible for X-linked juvenile retinoschisis, we have analyzed the expression, protein folding, disulfide-linked subunit assembly, intracellular localization, and secretion of wild-type retinoschisin, 15 Cys-to-Ser variants and 12 disease-linked mutants. Our studies, together with molecular modeling of the discoidin domain, identify Cys residues involved in intramolecular and intermolecular disulfide bonds essential for protein folding and subunit assembly. We show that misfolding of the discoidin domain, defective disulfide-linked subunit assembly, and inability of retinoschisin to insert into the endoplasmic reticulum membrane as part of the protein secretion process are three primary mechanisms responsible for the loss in the function of retinoschisin as a cell adhesion protein and the pathogenesis of X-linked juvenile retinoschisis.     

In silico Investigation of the Disease-Associated Retinoschisin C110Y and C219G Mutants

The juvenile X-linked retinoschisis (XLRS) is a retinal disease caused by mutations in the secretory protein, retinoschisin (RS1). Majority of the disease is resulted from single point mutations on the RS1 discoidin domain with cysteine mutations being related to some of the more severe cases of XLRS. Previous studies have indicated that two mutations (C110Y and C219G), which involve cysteines that form intramolecular disulfide bonds in the native discoidin domain, resulted in different oligomerization states of the proteins and did not correlate with the degree of protein stability as calculated by the change in folding free energy. Through homology modeling, bioinformatics predictions, molecular dynamics (MD) and docking simulations, we attempt to investigate the effects of these two mutations on the structure of the RS1 discoidin domain in relevance to the discrepancy found between structural stability and aggregation propensity. Based on our findings, this discrepancy can be explained by the ability of C110Y mutant to establish suitable modules for initiating amorphous aggregation and to expand the aggregating mass through predominantly hydrophobic interactions. The low capability of C219G mutant to oligomerize, on the other hand, may be due to its greater structural instability and lesser hydrophobic tendency, two properties that may be unsupportive of aggregation. The results, altogether, indicate that aggregation propensity in the RS1 C110Y mutant is dependent upon the formation of suitable aggregating substrates for propagation of aggregation and not directly related to or determined by overall structural instability. As for the wildtype protein, the binding specificity of the spikes for biological function and the formation of octameric structure are contributed by important loop interactions, as well as evolved structural and sequence-based properties that prevent aggregation.     

Characterization and purification of the discoidin domain-containing protein retinoschisin and its interaction with galactose

RS1, also known as retinoschisin, is an extracellular discoidin domain-containing protein that has been implicated in maintaining the cellular organization and synaptic structure of the vertebrate retina. Mutations in the gene encoding RS1 are responsible for X-linked retinoschisis, a retinal degenerative disease characterized by the splitting of the retinal cell layers and visual impairment. To better understand the role of RS1 in retinal cell biology and X-linked retinoschisis, we have studied the interaction of wild-type and mutant RS1 with various carbohydrates coupled to agarose supports. RS1 bound efficiently to galactose-agarose and to a lesser extent lactose-agarose, but not agarose, N-acetylgalactosamine-agarose, N-acetylglucosamine-agarose, mannose-agarose, or heparin-agarose. RS1 cysteine mutants (C59S/C223S and C59S/C223S/C40S) which prevent disulfide-linked octamer formation exhibited little if any binding to galactose-agarose. The disease-causing R141H mutant bound galactose-agarose at levels similar to that of wild-type RS1, whereas the R141S mutant resulted in a marked reduction in the level of galactose-agarose binding. RS1 bound to galactose-agarose could be effectively displaced by incubation with isopropyl beta- d-1-thiogalactopyranoside (IPTG). This property was used as a basis to develop an efficient purification procedure. Anion exchange and galactose affinity chromatography was used to purify RS1 from the culture media of stably transformed Sf21 insect cells that express and secrete RS1. This cell expression and protein purification method should prove useful in the isolation of RS1 for detailed structure-function studies.     

Retinoschisin (RS1), the protein encoded by the X-linked retinoschisis gene, is anchored to the surface of retinal photoreceptor and bipolar cells through its interactions with a Na/K ATPase-SARM1 complex

Retinoschisin or RS1 is a discoidin domain-containing protein encoded by the gene responsible for X-linked retinoschisis (XLRS), an early onset macular degeneration characterized by a splitting of the retina. Retinoschisin, expressed and secreted from photoreceptors and bipolar cells as a homo-octameric complex, associates with the surface of these cells where it serves to maintain the cellular organization of the retina and the photoreceptor-bipolar synaptic structure. To gain insight into the role of retinoschisin in retinal cell adhesion and the pathogenesis of XLRS, we have investigated membrane components in retinal extracts that interact with retinoschisin. Unlike the discoidin domain-containing blood coagulation proteins Factor V and Factor VIII, retinoschisin did not bind to phospholipids or retinal lipids reconstituted into unilamellar vesicles or immobilized on microtiter plates. Instead, co-immunoprecipitation studies together with mass spectrometric-based proteomics and Western blotting showed that retinoschisin is associated with a complex consisting of Na/K ATPase (alpha3, beta2 isoforms) and the sterile alpha and TIR motif-containing protein SARM1. Double labeling studies for immunofluorescence microscopy confirmed the co-localization of retinoschisin with Na/K ATPase and SARM1 in photoreceptors and bipolar cells of retina tissue. We conclude that retinoschisin binds to Na/K ATPase on photoreceptor and bipolar cells. This interaction may be part of a novel SARM1-mediated cell signaling pathway required for the maintenance of retinal cell organization and photoreceptor-bipolar synaptic structure.     

Effects of pathological mutations on the stability of a conserved amino acid triad in retinoschisin

A three-dimensional model has been calculated for the discoidin domain of retinoschisin (RS1), the protein involved in the X-linked juvenile retinoschisis. The model allows for a mapping of the pathological retinoschisis missense mutations and a rationale for the structural effects of an evolutionary conserved surface exposed triad (W122-R200-W163). Molecular dynamics simulations of the triad mutants models, together with ab initio energy calculations of the complexes corresponding to the triad show that the observed pathological mutations sensibly destabilize local interactions and the entire fold. Moreover the presented model reveals evidence of a putative site for membrane association.     

Mapping of epitopes in discoidin domain receptor 1 critical for collagen binding

The binding and activation of the discoidin domain receptor 1 by collagen has led to the conclusion that proteins from the extracellular matrix can directly induce receptor tyrosine kinase-mediated signaling cascades. A region in the extracellular domain of DDR1 homologous to the Dictyostelium discoideum protein discoidin-I is also present in the secreted human protein RS1. Mutations in RS1 cause retinoschisis, a genetic disorder characterized by ablation of the retina. By introducing point mutations into the discoidin domain of DDR1 at positions homologous to the retinoschisis mutations, ligand binding epitopes in the discoidin domain of DDR1 were mapped. Surprisingly, some residues only affected receptor phosphorylation, whereas others influenced both collagen-binding and receptor activation. Furthermore, two truncated DDR1 variants, lacking either the discoidin domain or the stalk region between the discoidin and transmembrane domain, were generated. We showed that (i) the discoidin domain was necessary and sufficient for collagen binding, (ii) only the region between discoidin and transmembrane domain was glycosylated, and (iii) the entire extracellular domain was essential for transmembrane signaling. Using these results, we were able to predict key sites in the collagen-binding epitope of DDR1 and to suggest a potential mechanism of signaling.     

Recurrent missense (R197C) and nonsense (Y89X) mutations in the XLRS1 gene in families with X-linked retinoschisis

Congenital retinoschisis (RS) is a hereditary eye disorder characterized by intraretinal schisis and central and peripheral retinal lesion. The gene responsible for the X-linked retinoschisis (XLRS1) has recently been isolated and found to contain mutations in affected members of several families. In this communication, two families with X-linked RS were analyzed for possible disease-causing mutations by polymerase chain reaction amplification of exons followed by DNA sequencing. Our analyses reveal a missense mutation at codon 197 in exon 6 and a nonsense mutation in exon-4 of XLRS1 gene. These changes resulted in the replacement of a highly conserved arginine by a cysteine residue and introduced a premature termination signal at codon 89, respectively. These mutations, which are transmitted through three generations, cosegregated with the disease, and are not found in the unaffected family members and 150 normal X-chromosomes, are likely to be pathogenic in these families.     

Exclusion of PPEF as the gene causing X-linked juvenile retinoschisis

X-linked juvenile retinoschisis (RS) is a progressive vitreoretinal degeneration localised in Xp22.1-p22.2. A human homologue of the retinal degeneration gene C (rdgC), a gene that in Drosophila melanogaster prevents light-induced retinal degeneration, was localised in the RS obligate gene region. We have tested the gene, designated PPEF in humans, as a candidate gene in RS patients using RT-PCR and the protein truncation test on RNA and SSCP on DNA. No mutations were identified, making it highly unlikely that PPEF is the gene implicated in RS. The data presented facilitate mutation analysis of the PPEF gene in other diseases which have been or will be localised to this region. Received: 20 May 1997 / Accepted: 30 July 1997     

CRX controls retinal expression of the X-linked juvenile retinoschisis (RS1) gene

X-linked juvenile retinoschisis is a heritable condition of the retina in males caused by mutations in the RS1 gene. Still, the cellular function and retina-specific expression of RS1 are poorly understood. To address the latter issue, we characterized the minimal promoter driving expression of RS1 in the retina. Binding site prediction, site-directed mutagenesis, and reporter assays suggest an essential role of two nearby cone-rod homeobox (CRX)-responsive elements (CRE) in the proximal −177/+32 RS1 promoter. Chromatin immunoprecipitation associates the RS1 promoter in vivo with CRX, the coactivators CBP, P300, GCN5 and acetylated histone H3. Transgenic Xenopus laevis expressing a green fluorescent protein (GFP) reporter under the control of RS1 promoter sequences show that the −177/+32 fragment drives GFP expression in photoreceptors and bipolar cells. Mutating either of the two conserved CRX binding sites results in strongly decreased RS1 expression. Despite the presence of sequence motifs in the promoter, NRL and NR2E3 appear not to be essential for RS1 expression. Together, our in vitro and in vivo results indicate that two CRE sites in the minimal RS1 promoter region control retinal RS1 expression and establish CRX as a key factor driving this expression.     

The RCK domain of the KtrAB K+ transporter: multiple conformations of an octameric ring

The KtrAB ion transporter is a complex of the KtrB membrane protein and KtrA, an RCK domain. RCK domains regulate eukaryotic and prokaryotic membrane proteins involved in K(+) transport. Conflicting functional models have proposed two different oligomeric arrangements for RCK domains, tetramer versus octamer. Our results for the KtrAB RCK domain clearly show an octamer in solution and in the crystal. We determined the structure of this protein in three different octameric ring conformations that resemble the RCK-domain octamer observed in the MthK potassium channel but show striking differences in size and symmetry. We present experimental evidence for the association between one RCK octameric ring and two KtrB membrane proteins. These results provide insights into the quaternary organization of the KtrAB transporter and its mechanism of activation and show that the RCK-domain octameric ring model is generally applicable to other ion-transport systems.     

Role of the N-terminal domain of the human DMC1 protein in octamer formation and DNA binding

The DMC1 protein, a eukaryotic homologue of RecA that shares significant amino acid identity with RAD51, exhibits two oligomeric DNA binding forms, an octameric ring and a helical filament. In the crystal structure of the octameric ring form, the DMC1 N-terminal domain (1-81 amino acid residues) was highly flexible, with multiple conformations. On the other hand, the N-terminal domain of Rad51 makes specific interactions with the neighboring ATPase domain in the helical filament structure. To gain insights into the functional role of the N-terminal domain of DMC1, we prepared a deletion mutant, DMC1-(82-340), that lacks the N-terminal 81 amino acid residues from the human DMC1 protein. Analytical ultracentrifugation experiments revealed that, whereas full-length DMC1 forms a octamer, DMC1-(82-340) is a heptamer. Furthermore, DNA binding experiments showed that DMC1-(82-340) was completely defective in both single-stranded and double-stranded DNA binding activities. Therefore, the N-terminal domain of DMC1 is required for the formation of the octamer, which may support the proper DNA binding activity of the DMC1 protein.     

Intraflagellar transport proteins 172, 80, 57, 54, 38, and 20 form a stable tubulin‐binding IFT‐B2 complex

Intraflagellar transport (IFT) relies on the IFT complex and is required for ciliogenesis. The IFT‐B complex consists of 9–10 stably associated core subunits and six “peripheral” subunits that were shown to dissociate from the core structure at moderate salt concentration. We purified the six “peripheral” IFT‐B subunits of Chlamydomonas reinhardtii as recombinant proteins and show that they form a stable complex independently of the IFT‐B core. We suggest a nomenclature of IFT‐B1 (core) and IFT‐B2 (peripheral) for the two IFT‐B subcomplexes. We demonstrate that IFT88, together with the N‐terminal domain of IFT52, is necessary to bridge the interaction between IFT‐B1 and B2. The crystal structure of IFT52N reveals highly conserved residues critical for IFT‐B1/IFT‐B2 complex formation. Furthermore, we show that of the three IFT‐B2 subunits containing a calponin homology (CH) domain (IFT38, 54, and 57), only IFT54 binds αβ‐tubulin as a potential IFT cargo, whereas the CH domains of IFT38 and IFT57 mediate the interaction with IFT80 and IFT172, respectively. Crystal structures of IFT54 CH domains reveal that tubulin binding is mediated by basic surface‐exposed residues.     

Linkage disequilibrium and physical mapping of X-linked juvenile retinoschisis.

X-linked juvenile retinoschisis (RS) is a recessively inherited disorder resulting in poor visual acuity. Affected males typically show retinal degeneration and intraretinal splitting. The prevalence of RS is 1:15,000-1:30,000. Elsewhere we have mapped the RS gene between the markers DXS43 and DXS274 in Xp22.1-p22.2. To narrow the RS region, we analyzed 31 Finnish RS families with the markers DXS418, DXS999, DXS7161, and DXS365 and a new polymorphic microsatellite marker, HYAT1. Multipoint linkage analysis allowed us to localize the RS gene between the markers DXS418 and DXS7161 (LOD score = 31.3). We have covered this region with nine YAC clones. On the basis of the sizes of the YACs, sequence-tagged site (STS) content mapping, and restriction mapping, the physical distance between DXS418 and DXS7161 is approximately 0.9 Mb. A total of five potential CpG islands could be identified. For haplotype analysis, eight additional Finnish RS families were analyzed with the markers DXS1195, DXS418, HYAT1, DXS999, DXS7161, and DXS365. On the basis of the linkage-disequilibrium data that were derived from the genetically isolated Finnish population, the critical region for RS could be narrowed to 0.2-0.3 cM, between the markers DXS418 and HYAT1.     

Analysis of the interaction mode between hyperthermophilic archaeal group II chaperonin and prefoldin using a platform of chaperonin oligomers of various subunit arrangements

Prefoldin is a co-chaperone that captures an unfolded protein substrate and transfers it to the group II chaperonin for completion of protein folding. Group II chaperonin of a hyperthermophilic archaeon, Thermococcus strain KS-1, interacts and cooperates with archaeal prefoldins. Although the interaction sites within chaperonin and prefoldin have been analyzed, the binding mode between jellyfish-like hexameric prefoldin and the double octameric ring group II chaperonin remains unclear. As prefoldin binds the chaperonin β subunit more strongly than the α subunit, we analyzed the binding mode between prefoldin and chaperonin in the context of Thermococcus group II chaperonin complexes of various subunit compositions and arrangements. The oligomers exhibited various affinities for prefoldins according to the number and order of subunits. Binding affinity increased with the number of Cpnβ subunits. Interestingly, chaperonin complexes containing two β subunits adjacently exhibited stronger affinities than other chaperonin complexes containing the same number of β subunits. The result suggests that all four β tentacles of prefoldin interact with the helical protrusions of CPN in the PFD–CPN complex as the previously proposed model that two adjacent PFD β subunits seem to interact with two CPN adjacent subunits.     

Crystal structure of the RuvA-RuvB complex: a structural basis for the Holliday junction migrating motor machinery

We present the X-ray structure of the RuvA-RuvB complex, which plays a crucial role in ATP-dependent branch migration. Two RuvA tetramers form the symmetric and closed octameric shell, where four RuvA domain IIIs spring out in the two opposite directions to be individually caught by a single RuvB. The binding of domain III deforms the protruding beta hairpin in the N-terminal domain of RuvB and thereby appears to induce a functional and less symmetric RuvB hexameric ring. The model of the RuvA-RuvB junction DNA ternary complex, constructed by fitting the X-ray structure into the averaged electron microscopic images of the RuvA-RuvB junction, appears to be more compatible with the branch migration mode of a fixed RuvA-RuvB interaction than with a rotational interaction mode.     

Exo84 and Sec5 are competitive regulatory Sec6/8 effectors to the RalA GTPase

The Sec6/8 complex, also known as the exocyst complex, is an octameric protein complex that has been implicated in tethering of secretory vesicles to specific regions on the plasma membrane. Two subunits of the Sec6/8 complex, Exo84 and Sec5, have recently been shown to be effector targets for active Ral GTPases. However, the mechanism by which Ral proteins regulate the Sec6/8 activities remains unclear. Here, we present the crystal structure of the Ral-binding domain of Exo84 in complex with active RalA. The structure reveals that the Exo84 Ral-binding domain adopts a pleckstrin homology domain fold, and that RalA interacts with Exo84 via an extended interface that includes both switch regions. Key residues of Exo84 and RalA were found that determine the specificity of the complex interactions; these interactions were confirmed by mutagenesis binding studies. Structural and biochemical data show that Exo84 and Sec5 competitively bind to active RalA. Taken together, these results further strengthen the proposed role of RalA-regulated assembly of the Sec6/8 complex.     

Biochemical and functional studies on the regulation of the Saccharomyces cerevisiae AMPK homolog SNF1

AMP-activated protein kinase (AMPK) is a master metabolic regulator for controlling cellular energy homeostasis. Its homolog in yeast, SNF1, is activated in response to glucose depletion and other stresses. The catalytic (α) subunit of AMPK/SNF1, Snf1 in yeast, contains a protein Ser/Thr kinase domain (KD), an auto-inhibitory domain (AID), and a region that mediates interactions with the two regulatory (β and γ) subunits. Previous studies suggested that Snf1 contains an additional segment, a regulatory sequence (RS, corresponding to residues 392-518), which may also have an important role in regulating the activity of the enzyme. The crystal structure of the heterotrimer core of Saccharomyces cerevisiae SNF1 showed interactions between a part of the RS (residues 460-498) and the γ subunit Snf4. Here we report biochemical and functional studies on the regulation of SNF1 by the RS. GST pulldown experiments demonstrate strong and direct interactions between residues 450-500 of the RS and the heterotrimer core, and single-site mutations in the RS-Snf4 interface can greatly reduce these interactions in vitro. On the other hand, functional studies appear to show only small effects of the RS-Snf4 interactions on the activity of SNF1 in vivo. This suggests that residues 450-500 may be constitutively associated with Snf4, and the remaining segments of the RS, as well as the AID, may be involved in regulating SNF1 activity.