A conserved inter-domain communication mechanism regulates the ATPase activity of the AAA-protein Drg1.

AAA-ATPases fulfil essential roles in different cellular pathways and often act in form of hexameric complexes. Interaction with pathway-specific substrate and adaptor proteins recruits them to their targets and modulates their catalytic activity. This substrate dependent regulation of ATP hydrolysis in the AAA-domains is mediated by a non-catalytic N-terminal domain. The exact mechanisms that transmit the signal from the N-domain and coordinate the individual AAA-domains in the hexameric complex are still the topic of intensive research. Here, we present the characterization of a novel mutant variant of the eukaryotic AAA-ATPase Drg1 that shows dysregulation of ATPase activity and altered interaction with Rlp24, its substrate in ribosome biogenesis. This defective regulation is the consequence of amino acid exchanges at the interface between the regulatory N-domain and the adjacent D1 AAA-domain. The effects caused by these mutations strongly resemble those of pathological mutations of the AAA-ATPase p97 which cause the hereditary proteinopathy IBMPFD (inclusion body myopathy associated with Paget's disease of the bone and frontotemporal dementia). Our results therefore suggest well conserved mechanisms of regulation between structurally, but not functionally related members of the AAA-family.

The drug diazaborine blocks ribosome biogenesis by inhibiting the AAA-ATPase Drg1.

The drug diazaborine is the only known inhibitor of ribosome biogenesis and specifically blocks large subunit formation in eukaryotic cells. However, the target of this drug and the mechanism of inhibition were unknown. Here we identify the AAA-ATPase Drg1 as a target of diazaborine. Inhibitor binding into the second AAA domain of Drg1 requires ATP loading and results in inhibition of ATP hydrolysis in this site. As a consequence the physiological activity of Drg1, i.e. the release of Rlp24 from pre-60S particles, is blocked, and further progression of cytoplasmic preribosome maturation is prevented. Our results identify the first target of an inhibitor of ribosome biogenesis and provide the mechanism of inhibition of a key step in large ribosomal subunit formation.

Phenylalanine 368 of multidrug resistance-associated protein 4 (MRP4/ABCC4) plays a crucial role in substrate-specific transport activity.

Multidrug resistance-associated protein 4 (MRP4) is a membrane transporter that mediates the cellular efflux of a wide range of anionic drugs and endogenous molecules. MRP4 transport can influence the pharmacokinetics of drugs and their metabolites, therefore more knowledge about the molecular determinants important for its transport function would be of relevance. Here, we substituted amino acids Phe(368), Trp(995), and Arg(998) with conservative or non-conservative residues, and determined the effect on transport of the model substrates estradiol 17-ß-d-glucuronide (E(2)17ßG), cyclic guanosine monophosphate (cGMP), methotrexate (MTX), and folic acid into membrane vesicles isolated from baculovirus transduced HEK293 cells overexpressing the mutant MRP4 proteins. This revealed that all Arg(998) mutations appeared to be deleterious, whereas the effect of a Phe(368) or Trp(995) replacement was dependent on the amino acid introduced and the substrate studied. Substitution of Phe(368) with Trp (F368W) induced a gain-of-function of E(2)17ßG transport and a loss-of-function of MTX transport, which could not be attributed to an altered substrate binding. Moreover, we did not observe any modification in ATP or ADP handling for F368W. These results, in combination with docking of substrates in a homology model of MRP4 in the inward- and outward-facing conformation, suggest that Phe(368) and Trp(995) do not play an important role in the initial binding of substrates. They, however, might interact with the substrates during rearrangement of helixes for substrate translocation, funneling the substrates to the exit site in the outward-facing conformation.

A series of PDB related databases for everyday needs.

The Protein Data Bank (PDB) is the world-wide repository of macromolecular structure information. We present a series of databases that run parallel to the PDB. Each database holds one entry, if possible, for each PDB entry. DSSP holds the secondary structure of the proteins. PDBREPORT holds reports on the structure quality and lists errors. HSSP holds a multiple sequence alignment for all proteins. The PDBFINDER holds easy to parse summaries of the PDB file content, augmented with essentials from the other systems. PDB_REDO holds re-refined, and often improved, copies of all structures solved by X-ray. WHY_NOT summarizes why certain files could not be produced. All these systems are updated weekly. The data sets can be used for the analysis of properties of protein structures in areas ranging from structural genomics, to cancer biology and protein design.

Folding defects in P-type ATP 8B1 associated with hereditary cholestasis are ameliorated by 4-phenylbutyrate.

UNLABELLED: Deficiency in P-type ATP8B1 is a severe and clinically highly variable hereditary disorder that is primarily characterized by intrahepatic cholestasis. It presents either as a progressive (progressive familial intrahepatic cholestasis type 1 [PFIC1]) or intermittent (benign recurrent intrahepatic cholestasis type 1 [BRIC1]) disease. ATP8B1 deficiency is caused by autosomal recessive mutations in the gene encoding ATP8B1, a putative aminophospholipid-translocating P-type adenosine triphosphatase. The exact pathogenesis of the disease is elusive, and no effective pharmacological therapy is currently available. Here, the molecular consequences of six distinct ATP8B1 missense mutations (p.L127P, p.G308V, p.D454G, p.D554N, p.I661T, and p.G1040R) and one nonsense mutation (p.R1164X) associated with PFIC1 and/or BRIC1 were systematically characterized. Except for the p.L127P mutation, all mutations resulted in markedly reduced ATP8B1 protein expression, whereas messenger RNA expression was unaffected. Five of seven mutations resulted in (partial) retention of ATP8B1 in the endoplasmic reticulum. Reduced protein expression was partially restored by culturing the cells at 30 degrees C and by treatment with proteasomal inhibitors, indicating protein misfolding and subsequent proteosomal degradation. Protein misfolding was corroborated by predicting the consequences of most mutations onto a homology model of ATP8B1. Treatment with 4-phenylbutyrate, a clinically approved pharmacological chaperone, partially restored defects in expression and localization of ATP8B1 substitutions G308V, D454G, D554N, and in particular I661T, which is the most frequently identified mutation in BRIC1. CONCLUSION: A surprisingly large proportion of ATP8B1 mutations resulted in aberrant folding and decreased expression at the plasma membrane. These effects were partially restored by treatment with 4-phenylbutyrate. We propose that treatment with pharmacological chaperones may represent an effective therapeutic strategy to ameliorate the recurrent attacks of cholestasis in patients with intermittent (BRIC1) disease.

Reduced expression of ATP7B affected by Wilson disease-causing mutations is rescued by pharmacological folding chaperones 4-phenylbutyrate and curcumin.

UNLABELLED: Wilson disease (WD) is an autosomal recessive copper overload disorder of the liver and basal ganglia. WD is caused by mutations in the gene encoding ATP7B, a protein localized to the trans-Golgi network that primarily facilitates hepatic copper excretion. Current treatment comprises reduction of circulating copper by zinc supplementation or copper chelation. Despite treatment, a significant number of patients have neurological deterioration. The aim of this study was to investigate the possibility that defects arising from some WD mutations are ameliorated by drug treatment aimed at improvement of protein folding and restoration of protein function. This necessitated systematic characterization of the molecular consequences of distinct ATP7B missense mutations associated with WD. With the exception of p.S1363F, all mutations tested (p.G85V, p.R778L, p.H1069Q, p.C1104F, p.V1262F, p.G1343V, and p.S1363F) resulted in reduced ATP7B protein expression, whereas messenger RNA abundance was unaffected. Retention of mutant ATP7B in the endoplasmic reticulum, increased protein expression, and normalization of localization after culturing cells at 30 degrees C, and homology modeling suggested that these proteins were misfolded. Four distinct mutations exhibited residual copper export capacity, whereas other mutations resulted in complete disruption of copper export by ATP7B. Treatment with pharmacological chaperones 4-phenylbutyrate (4-PBA) and curcumin, a clinically approved compound, partially restored protein expression of most ATP7B mutants. CONCLUSION: These findings might enable novel treatment strategies in WD by directly enhancing the protein expression of mutant ATP7B with residual copper export activity. 1795.).

MYO15A (DFNB3) mutations in Turkish hearing loss families and functional modeling of a novel motor domain mutation.

Myosin XVA is an unconventional myosin which has been implicated in autosomal recessive nonsyndromic hearing impairment (ARNSHI) in humans. In Myo15A mouse models, vestibular dysfunction accompanies the autosomal recessive hearing loss. Genomewide homozygosity mapping and subsequent fine mapping in two Turkish families with ARNSHI revealed significant linkage to a critical interval harboring a known deafness gene MYO15A on chromosome 17p13.1-17q11.2. Subsequent sequencing of the MYO15A gene led to the identification of a novel missense mutation, c.5492G-->T (p.Gly1831Val) and a novel splice site mutation, c.8968-1G-->C. These mutations were not detected in additional 64 unrelated ARNSHI index patients and in 230 Turkish control chromosomes. Gly1831 is a conserved residue located in the motor domains of the different classes of myosins of different species. Molecular modeling of the motor head domain of the human myosin XVa protein suggests that the Gly1831Val mutation inhibits the powerstroke by reducing backbone flexibility and weakening the hydrophobic interactions necessary for signal transmission to the converter domain.

GTP-Ras disrupts the intramolecular complex of C1 and RA domains of Nore1.

The novel Ras effector mNore1, capable of inducing apoptosis, is a multidomain protein. It comprises a C1 domain homologous to PKC and an RA domain similar to the Ras effectors AF-6 and RalGDS. Here, we determine the affinity of these two domains to the active forms of Ras and Rap1 using isothermal calorimetric titration. The interaction of Ras/Rap1-GTP with the RA domain of mNore1 is weakened significantly by direct binding of the C1 domain to the RA domain. In order to analyze this observation in atomic detail, we solved the C1 solution structure by NMR. By determining chemical shifts and relaxation rates, we can show an intramolecular complex of C1-RA. GTP-Ras titration and binding to RA disrupts this complex and displaces the C1 domain. Once the C1 domain tumbles freely in solution, a lipid binding interface becomes accessible. Furthermore, we provide evidence of phosphatidylinositol 3-phosphate binding of the free C1 domain.

Definition of a new information-based per-residue quality parameter.

For biomolecular NMR structures typically only a poor correspondence is observed between statistics derived from the experimental input data and structural quality indicators obtained from the structure ensembles. Here, we investigate the relationship between the amount of available NMR data and structure quality. By generating datasets with a predetermined information content and evaluating the quality of the resulting structure ensembles we show that there is, in contrast to previous findings, a linear relation between the information contained in experimental data and structural quality. From this relation, a new quality parameter is derived that provides direct insight, on a per-residue basis, into the extent to which structural quality is governed by the experimental input data.

Reconstruction of the complete ouabain-binding pocket of Na,K-ATPase in gastric H,K-ATPase by substitution of only seven amino acids.

Although cardiac glycosides have been used as drugs for more than 2 centuries and their primary target, the sodium pump (Na,K-ATPase), has already been known for 4 decades, their exact binding site is still elusive. In our efforts to define the molecular basis of digitalis glycosides binding we started from the fact that a closely related enzyme, the gastric H,K-ATPase, does not bind glycosides like ouabain. Previously, we showed that a chimera of these two enzymes, in which only the M3-M4 and M5-M6 hairpins were of Na,K-ATPase, bound ouabain with high affinity (Koenderink, J. B., Hermsen, H. P. H., Swarts, H. G. P., Willems, P. H. G. M., and De Pont, J. J. H. H. M. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 11209-11214). We also demonstrated that only three amino acids (Phe(783), Thr(797), and Asp(804)) present in the M5-M6 hairpin of Na,K-ATPase were sufficient to confer high affinity ouabain binding to a chimera which contained in addition the M3-M4 hairpin of Na,K-ATPase (Qiu, L. Y., Koenderink, J. B., Swarts, H. G., Willems, P. H., and De Pont, J. J. H. H. M. (2003) J. Biol. Chem. 278, 47240-47244). To further pinpoint the ouabain-binding site here we used a chimera-based loss-of-function strategy and identified four amino acids (Glu(312), Val(314), Ile(315), Gly(319)), all present in M4, as being important for ouabain binding. In a final gain-of-function study we showed that a gastric H,K-ATPase that contained Glu(312), Val(314), Ile(315), Gly(319), Phe(783), Thr(797), and Asp(804) of Na,K-ATPase bound ouabain with the same affinity as the native enzyme. Based on the E(2)P crystal structure of Ca(2+)-ATPase we constructed a homology model for the ouabain-binding site of Na,K-ATPase involving all seven amino acids as well as several earlier postulated amino acids.

Asn792 participates in the hydrogen bond network around the K+-binding pocket of gastric H,K-ATPase.

Asn792 present in M5 of gastric H,K-ATPase is highly conserved within the P-type ATPase family. A direct role in K+ binding was postulated for Na,K-ATPase but was not found in a recent model for gastric H,K-ATPase (Koenderink, J. B., Swarts, H. G. P., Willems, P. H. G. M., Krieger, E., and De Pont, J. J. H. H. M. (2004) J. Biol. Chem. 279, 16417-16424). Therefore, its role in K+ binding and E1/E2 conformational equilibrium in gastric H,K-ATPase was studied by site-directed mutagenesis and expression in Sf9 cells. N792Q and N792A, but not N792D and N792E, had a markedly reduced K+ affinity in both the ATPase and dephosphorylation reactions. In addition, N792A shifted the conformational equilibrium to the E1 form. In double mutants, the effect of N792A on K+ sensitivity was overruled by either E820Q (K(+)-independent activity) or E343D (no dephosphorylation activity). Models were made for the mutants based on the E2 structure of Ca(2+)-ATPase. In the wild-type model the acid amide group of Asn792 has hydrogen bridges to Lys791, Ala339, and Val341. Comparison of the effects of the various mutants suggests that the hydrogen bridge between the carbonyl oxygen of Asn792 and the amino group of Lys791 is essential for the K+ sensitivity and the E2 preference of wild-type enzyme. Moreover, there was a high positive correlation (r = 0.98) between the in silico calculated energy difference of the E2 form (mutants versus wild type) and the experimentally measured IC50 values for vanadate, which reflects the direction of the E2<-->E1 conformational equilibrium. These data strongly support the validity of the model in which Asn792 participates in the hydrogen bond network around the K(+)-binding pocket.

Identification and molecular modelling of a mutation in the motor head domain of myosin VIIA in a family with autosomal dominant hearing impairment (DFNA11).

Myosin VIIA is an unconventional myosin that has been implicated in Usher syndrome type 1B, atypical Usher syndrome, non-syndromic autosomal recessive hearing impairment (DFNB2) and autosomal dominant hearing impairment (DFNA11). Here, we present a family with non-syndromic autosomal dominant hearing impairment that clinically resembles the previously published DFNA11 family. The affected family members show a flat audiogram at young ages and only modest progression, most clearly at the high frequencies. In addition, they suffer from minor vestibular symptoms. Linkage analysis yielded a maximum two-point lodscore of 3.43 for marker D11S937 located within 1 cM of the myosin VIIA gene. The myosin VIIA gene was sequenced and 11 nucleotide variations were found. Ten nucleotide changes represent benign intronic variants, silent exon mutations or non-pathologic amino acid substitutions. One variant, a c.1373A-->T transversion that is heterozygously present in all affected family members and absent in 300 healthy individuals, is predicted to result in an Asn458Ile amino acid substitution. Asn458 is located in a region of the myosin VIIA motor domain that is highly conserved in different classes of myosins and in myosins of different species. To evaluate whether the Asn458Ile mutation was indeed responsible for the hearing impairment, a molecular model of myosin VIIA was built based on the known structure of the myosin II heavy chain from Dictyostelium discoideum. In this model, conformational changes in the protein caused by the amino acid substitution Asn458Ile are predicted to disrupt ATP/ADP binding and impair the myosin power-stroke, which would have a severe effect on the function of the myosin VIIA protein.

Mutations in the DJ-1 gene associated with autosomal recessive early-onset parkinsonism.

The DJ-1 gene encodes a ubiquitous, highly conserved protein. Here, we show that DJ-1 mutations are associated with PARK7, a monogenic form of human parkinsonism. The function of the DJ-1 protein remains unknown, but evidence suggests its involvement in the oxidative stress response. Our findings indicate that loss of DJ-1 function leads to neurodegeneration. Elucidating the physiological role of DJ-1 protein may promote understanding of the mechanisms of brain neuronal maintenance and pathogenesis of Parkinson's disease.

Gain-of-function mutation in ADULT syndrome reveals the presence of a second transactivation domain in p63.

The transcriptional co-activator p63 is of crucial importance for correct development of the limbs, ectodermal appendages (skin, nails, teeth, hair, glands), lip and palate. Mutations in the p63 gene are found in a number of human syndromes, including ectrodactyly-ectodermal dysplasia-cleft lip/palate (EEC) syndrome, limb-mammary syndrome (LMS), Hay-Wells syndrome and in non-syndromic split-hand/split-foot malformation (SHFM). Each syndrome has a specific pattern of mutations with different functional effects in in vitro functional assays. We report a mutation R298Q in acro-dermato-ungual-lacrimal-tooth (ADULT) syndrome, another EEC-like condition. The mutation is located in the DNA binding domain of p63, which harbors almost all EEC associated mutations. However, unlike mutations in EEC syndrome, the R298Q ADULT syndrome mutation does not impair DNA binding. Rather, the mutation confers novel transcription activation capacity on the DeltaN-p63gamma isoform, which normally does not possess such activity. These results confirm that ADULT syndrome is a clinically as well as molecularly distinct member of the expanding p63 mutation family of human malformation syndromes. Our results further show that p63 contains a second transactivation domain which is normally repressed and can become activated by mutations in the DNA binding domain of p63.

Identification of different binding sites in the dendritic cell-specific receptor DC-SIGN for intercellular adhesion molecule 3 and HIV-1.

The novel dendritic cell (DC)-specific human immunodeficiency virus type 1 (HIV-1) receptor DC-SIGN plays a key role in the dissemination of HIV-1 by DC. DC-SIGN is thought to capture HIV-1 at mucosal sites of entry, facilitating transport to lymphoid tissues, where DC-SIGN efficiently transmits HIV-1 to T cells. DC-SIGN is also important in the initiation of immune responses by regulating DC-T cell interactions through intercellular adhesion molecule 3 (ICAM-3). We have characterized the mechanism of ligand binding by DC-SIGN and identified the crucial amino acids involved in this process. Strikingly, the HIV-1 gp120 binding site in DC-SIGN is different from that of ICAM-3, consistent with the observation that glycosylation of gp120, in contrast to ICAM-3, is not crucial to the interaction with DC-SIGN. A specific mutation in DC-SIGN abrogated ICAM-3 binding, whereas the HIV-1 gp120 interaction was unaffected. This DC-SIGN mutant captured HIV-1 and infected T cells in trans as efficiently as wild-type DC-SIGN, demonstrating that ICAM-3 binding is not necessary for HIV-1 transmission. This study provides a basis for the design of drugs that inhibit or alter interactions of DC-SIGN with gp120 but not with ICAM-3 or vice versa and that have a therapeutic value in immunological diseases and/or HIV-1 infections.