Molecular simulation studies of human coagulation factor VIII C domain-mediated membrane binding
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The C-terminal C domains of activated coagulation factor VIII (FVIIIa) are essential to membrane binding of this crucial coagulation cofactor protein. To provide an overall membrane binding mechanism for FVIII, we performed simulations of membrane binding through coarse-grained molecular dynamics simulations of the C1 and C2 domain, and the combined C-domains (C1+C2). We found that the C1 and C2 domain have different membrane binding properties. The C1 domain uses hydrophobic spikes 3 and 4, of its total of four spikes, as major loops to bind the membrane, whereas all four of its hydrophobic loops of the C2 domain appear essential for membrane binding. Interestingly, in the C1+C2 system, we observed cooperative binding of the C1 and C2 domains such that all four C2 domain spikes bound first, after which all four loops of the C1 domain inserted into the membrane, while the net binding energy was higher than that of the sum of the isolated C domains. Several residues, mutations of which are known to cause haemophilia A, were identified as key residues for membrane binding. In addition to these known residues, we identified residues from the C1 and C2 domains, which are involved in the membrane binding process, that have not been reported before as a cause for haemophilia A, but which contribute to overall membrane binding and which are likely candidates for novel causative missense mutations in haemophilia A.Keywords:
C2 domain
Ternary complex
Protein A
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C2 domain
Factor V
Prothrombinase
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Protein kinase C is specifically activated by binding two membrane lipids: the second messenger, diacylglycerol, and the amino phospholipid, phosphatidylserine. This binding provides the energy to release an autoinhibitory pseudosubstrate from the active site. Interaction with these lipids recruits the enzyme to the membrane by engaging two membrane-targeting modules: the C1 domain (present as a tandem repeat in most protein kinase Cs) and the C2 domain. Here we dissect the contribution of each domain in recruiting protein kinase C βII to membranes. Binding analyses of recombinant domains reveal that the C2 domain binds anionic lipids in a Ca2+-dependent, but diacylglycerol-independent, manner, with little selectivity for phospholipid headgroup beyond the requirement for negative charge. The C1B domain binds membranes in a diacylglycerol/phorbol ester-dependent, but Ca2+-independent manner. Like the C2 domain, the C1B domain preferentially binds anionic lipids. However, in striking contrast to the C2 domain, the C1B domain binds phosphatidylserine with an order of magnitude higher affinity than other anionic lipids. This preference for phosphatidylserine is, like that of the full-length protein, stereoselective for sn-1,2-phosphatidyl-l-serine. Quantitative analysis of binding constants of individual domains and that of full-length protein reveals that the full-length protein binds membranes with lower affinity than expected based on the binding affinity of isolated domains. In addition to entropic and steric considerations, the difference in binding energy may reflect the energy required to expel the pseudosubstrate from the substrate binding cavity. This study establishes that each module is an independent membrane-targeting module with each, independently of the other, containing determinants for membrane recognition. The presence of each of these modules, separately, in a number of other signaling proteins epitomizes the use of these modules as discreet membrane targets.
C2 domain
HAMP domain
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In vitro protein binding assays identified two distinct calmodulin (CaM) binding sites within the NH2-terminal 30-kDa domain of erythrocyte protein 4.1 (4.1R): a Ca2+-independent binding site (A264KKLWKVCVEHHTFFRL) and a Ca2+-dependent binding site (A181KKLSMYGVDLHKAKDL). Synthetic peptides corresponding to these sequences bound CaM in vitro; conversely, deletion of these peptides from a 30-kDa construct reduced binding to CaM. Thus, 4.1R is a unique CaM-binding protein in that it has distinct Ca2+-dependent and Ca2+-independent high affinity CaM binding sites. CaM bound to 4.1R at a stoichiometry of 1:1 both in the presence and absence of Ca2+, implying that one CaM molecule binds to two distinct sites in the same molecule of 4.1R. Interactions of 4.1R with membrane proteins such as band 3 is regulated by Ca2+ and CaM. While the intrinsic affinity of the 30-kDa domain for the cytoplasmic tail of erythrocyte membrane band 3 was not altered by elimination of one or both CaM binding sites, the ability of Ca2+/CaM to down-regulate 4.1R-band 3 interaction was abrogated by such deletions. Thus, regulation of protein 4.1 binding to membrane proteins by Ca2+ and CaM requires binding of CaM to both Ca2+-independent and Ca2+-dependent sites in protein 4.1.
Band 3
EF hand
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C2 domain
Factor IXa
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Human serum albumin
Digitoxin
Binding constant
Serum Albumin
Antiinflammatory drug
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Rat alpha-foetoprotein (alpha-FP) strongly binds the drugs warfarin and phenylbutazone, as does albumin; however, the binding sites for the two drugs seemed to be different. This possibility and the specificity of this/these drug-binding site(s) of rat alpha-FP were investigated by competitive protein-binding experiments with a variety of drugs, representing different pharmacological groups, and biomolecules that are strongly bound by the foetal protein and that are suspected to play a specific role during foetal development. The binding mechanisms were further investigated by using comparisons between computer-derived theoretical displacement curves and experimental points in order to distinguish different possible binding models. The results indicate: that warfarin and phenylbutazone are bound at two distinct sites on rat alpha-FP and that a negative modulatory effect is exerted between the two sites; that the degree of specificity of these two drug-binding sites is different, since the warfarin-binding site appears to be specific for the binding of coumarinic and anthranilic drugs whereas that for phenylbutazone is able to bind substances of very varied chemical structure and is more hydrophobic; that the phenylbutazone-binding site is the site that binds oestrogens that thyroid hormones and, probably, fatty acids and bilirubin are bound at (an)other site(s) but exert negative modulatory effects on phenylbutazone binding. The nature of the different binding areas of rat alpha-FP is compared with that of those already proposed for albumin. The potential risks of toxicity of such interactions between drugs and/or biomolecules on foetal development are also discussed.
A-site
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The C2 domain was originally defined as a homologous domain to the C2 regulatory region of Ca2+-dependent protein kinase C and has been identified in more than 50 different signaling molecules. The original C2 domain of protein kinase Cα functions as a Ca2+ binding module, and the Ca2+ binding to the C2 domain allows translocation of proteins to phospholipid membranes. By contrast, however, some C2 domains do not exhibit Ca2+ binding activity because of amino acid substitutions at Ca2+-binding sites, and their physiological meanings remain largely unknown. In this study, we discovered an unexpected function of the Ca2+-independent C2A domain of double C2 protein γ (Doc2γ) in nuclear localization. Deletion and mutation analyses revealed that the putative Ca2+ binding loop 3 of Doc2γ contains six Arg residues (177RLRRRRR183) and that this basic cluster is both necessary and sufficient for nuclear localization of Doc2γ. Because of the presence of the basic cluster, the C2A domain of Doc2γ did not show Ca2+-dependent phospholipid binding activity. Our findings indicate that by changing the nature of the putative Ca2+ binding loops the C2 domain has more diversified function in cellular signaling than a simple Ca2+ binding motif. The C2 domain was originally defined as a homologous domain to the C2 regulatory region of Ca2+-dependent protein kinase C and has been identified in more than 50 different signaling molecules. The original C2 domain of protein kinase Cα functions as a Ca2+ binding module, and the Ca2+ binding to the C2 domain allows translocation of proteins to phospholipid membranes. By contrast, however, some C2 domains do not exhibit Ca2+ binding activity because of amino acid substitutions at Ca2+-binding sites, and their physiological meanings remain largely unknown. In this study, we discovered an unexpected function of the Ca2+-independent C2A domain of double C2 protein γ (Doc2γ) in nuclear localization. Deletion and mutation analyses revealed that the putative Ca2+ binding loop 3 of Doc2γ contains six Arg residues (177RLRRRRR183) and that this basic cluster is both necessary and sufficient for nuclear localization of Doc2γ. Because of the presence of the basic cluster, the C2A domain of Doc2γ did not show Ca2+-dependent phospholipid binding activity. Our findings indicate that by changing the nature of the putative Ca2+ binding loops the C2 domain has more diversified function in cellular signaling than a simple Ca2+ binding motif. synaptotagmin(s) carboxyl-terminal type double C2 protein glutathione S-transferase Munc13–1 interacting domain phosphatidylcholine phosphatidylserine 4,6-diamidino-2-phenylindole polymerase chain reaction The C2 domain is a Ca2+ binding motif that consists of ∼130 amino acids, and it has been identified in various signaling molecules including protein kinases, lipid modification enzymes, GTPase-activating proteins, ubiquitination enzymes, and proteins involved in vesicular trafficking (reviewed in Refs. 1Ponting C.P. Parker P.J. Protein Sci. 1996; 5: 162-166Crossref PubMed Scopus (155) Google Scholar and 2Nalefski E.A. Falke J.J. Protein Sci. 1996; 5: 2375-2390Crossref PubMed Scopus (689) Google Scholar). The C2 domain was originally defined as a homologous domain to the C2 regulatory region of mammalian Ca2+-dependent protein kinase C isoforms α, β, and γ (reviewed in Ref. 3Nishizuka Y. Nature. 1998; 334: 661-665Crossref Scopus (3535) Google Scholar). The C2 domains are composed of a common eight-stranded antiparallel β-sandwich consisting of four-stranded β-sheets, although their structures have been classified into two groups based on their topology (e.g. synaptotagmin I C2A domain with type I topology and phospholipase C δ1 C2 domain with type II topology) (2Nalefski E.A. Falke J.J. Protein Sci. 1996; 5: 2375-2390Crossref PubMed Scopus (689) Google Scholar,4Sutton R.B. Davletov B.A. Berghuis A.M. Südhof T.C. Sprang S.R. Cell. 1995; 80: 929-938Abstract Full Text PDF PubMed Scopus (605) Google Scholar, 5Essen L.O. Perisic O. Cheung R. Katan M. Williams R.L. Nature. 1996; 380: 595-602Crossref PubMed Scopus (516) Google Scholar). Three flexible loops protrude from the tip of the β-sandwich structure, and some of them are involved in Ca2+ binding (4Sutton R.B. Davletov B.A. Berghuis A.M. Südhof T.C. Sprang S.R. Cell. 1995; 80: 929-938Abstract Full Text PDF PubMed Scopus (605) Google Scholar, 5Essen L.O. Perisic O. Cheung R. Katan M. Williams R.L. Nature. 1996; 380: 595-602Crossref PubMed Scopus (516) Google Scholar). The Ca2+ binding allows interaction of the C2 domain with phospholipids to enable translocation of proteins to phospholipid membranes (2Nalefski E.A. Falke J.J. Protein Sci. 1996; 5: 2375-2390Crossref PubMed Scopus (689) Google Scholar). The role of the C2 domain is not limited to the phospholipid membrane interaction sites and has been shown to be a Ca2+-dependent and -independent protein interaction site. For instance, the synaptotagmin I (Syt I)1 C2 domain, one of the best characterized C2 domains essential for neurotransmitter release (reviewed in Refs. 6Südhof T.C. Rizo J. Neuron. 1996; 17: 379-388Abstract Full Text Full Text PDF PubMed Scopus (396) Google Scholar, 7Fukuda M. Mikoshiba K. Bioessays. 1997; 19: 593-603Crossref PubMed Scopus (89) Google Scholar, 8Schiavo G. Osborne S.L. Sgouros J.G. Biochem. Biophys. Res. Commun. 1998; 248: 1-8Crossref PubMed Scopus (99) Google Scholar, 9Marquèze B. Berton F. Seagar M. Biochimie ( Paris ). 2000; 82: 409-420Crossref PubMed Scopus (90) Google Scholar), has been shown to interact with negatively charged phospholipids (10Davletov B.A. Südhof T.C. J. Biol. Chem. 1993; 268: 26386-26390Abstract Full Text PDF PubMed Google Scholar, 11Chapman E.R. Jahn R. J. Biol. Chem. 1994; 269: 5735-5741Abstract Full Text PDF PubMed Google Scholar, 12Fukuda M. Aruga J. Niinobe M. Aimoto S. Mikoshiba K. J. Biol. Chem. 1994; 269: 29206-29211Abstract Full Text PDF PubMed Google Scholar, 13Fukuda M. Kojima T. Mikoshiba K. J. Biol. Chem. 1996; 271: 8430-8434Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar), syntaxin (14Li C. Ullrich B. Zhang J.Z. Anderson R.G.W. Brose N. Südhof T.C. Nature. 1995; 375: 594-599Crossref PubMed Scopus (541) Google Scholar), and Syt I itself in a Ca2+-dependent manner (15Sugita S. Hata Y. Südhof T.C. J. Biol. Chem. 1996; 271: 1262-1265Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 16Damer C.K. Creutz C.E. J. Neurochem. 1996; 67: 1661-1668Crossref PubMed Scopus (51) Google Scholar, 17Chapman E.R. Desai R.C. Davis A.F. Tornehl C.K. J. Biol. Chem. 1998; 273: 32966-32972Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar, 18Osborne S.L. Herreros J. Bastiaens P.I.H. Schiavo G. J. Biol. Chem. 1999; 274: 59-66Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 19Fukuda M. Mikoshiba K. J. Biol. Chem. 2000; 275: 28180-28185Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 20Fukuda M. Mikoshiba K. J. Biochem. ( Tokyo ). 2000; 128: 637-645Crossref PubMed Scopus (34) Google Scholar). In addition, the Syt I C2B domain binds inositol polyphosphates (21Fukuda M. Kojima T. Aruga J. Niinobe M. Mikoshiba K. J. Biol. Chem. 1995; 270: 26523-26527Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 22Ibata K. Fukuda M. Mikoshiba K. J. Biol. Chem. 1998; 273: 12267-12273Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar), clathrin assembly protein, AP-2 (23Zhang J.Z. Davletov B.A. Südhof T.C. Anderson R.G.W. Cell. 1994; 78: 751-760Abstract Full Text PDF PubMed Scopus (433) Google Scholar), SV2 (24Schivell A.E. Batchelor R.H. Bajjalieh S.M. J. Biol. Chem. 1996; 271: 27770-27775Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar), β-SNAP (25Schiavo G. Gmachl M.J. Stenbeck G. Söllner T.H. Rothman J.E. Nature. 1995; 378: 733-736Crossref PubMed Scopus (157) Google Scholar), SNAP25 (26Schiavo G. Stenbeck G. Rothman J.E. Söllner T.H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 997-1001Crossref PubMed Scopus (258) Google Scholar), Ca2+ channels (27Sheng Z.H. Yokoyama C.T. Catterall W.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5405-5410Crossref PubMed Scopus (162) Google Scholar), and SYNCRIP (Syt-binding, cytoplasmicRNA-interacting protein) (28Mizutani A. Fukuda M. Ibata K. Shiraishi Y. Mikoshiba K. J. Biol. Chem. 2000; 275: 9823-9831Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar), irrespective of the presence of Ca2+. Furthermore, some Syt isoforms fail to exhibit Ca2+ binding because of amino acid substitutions (mutation of the Glu or Asp residue involved in Ca2+ binding) in the putative Ca2+ binding loops (14Li C. Ullrich B. Zhang J.Z. Anderson R.G.W. Brose N. Südhof T.C. Nature. 1995; 375: 594-599Crossref PubMed Scopus (541) Google Scholar, 29Fukuda M. Mikoshiba K. Biochem. J. 2001; 354: 249-257Crossref PubMed Scopus (39) Google Scholar). However, the function of the Ca2+-independent type of C2 domains remains largely unknown. In this paper, we report the discovery of an unexpected function of the C2A domain of a third isoform of double C2 protein that contains a C2A domain and a C2B domain (Doc2γ; see Fig. 1 A) in nuclear localization (30Fukuda M. Mikoshiba K. Biochem. Biophys. Res. Commun. 2000; 276: 626-632Crossref PubMed Scopus (34) Google Scholar, 31Duncan R.R. Shipston M.J. Chow R.H. Biochimie ( Paris ). 2000; 82: 421-426Crossref PubMed Scopus (41) Google Scholar). Unlike other members of the Doc2 family, the C2A domain of Doc2γ lacks Ca2+-dependent phospholipid binding activity, probably because of the amino acid substitutions of the key amino acids (Glu or Asp) responsible for Ca2+ binding (30Fukuda M. Mikoshiba K. Biochem. Biophys. Res. Commun. 2000; 276: 626-632Crossref PubMed Scopus (34) Google Scholar, 32Kojima T. Fukuda M. Aruga J. Mikoshiba K. J. Biochem. ( Tokyo ). 1996; 120: 671-676Crossref PubMed Scopus (45) Google Scholar, 33Fukuda M. Kojima T. Mikoshiba K. Biochem. J. 1997; 323: 421-425Crossref PubMed Scopus (39) Google Scholar, 34Orita S. Sasaki T. Naito A. Komuro R. Ohtsuka T. Maeda M. Suzuki H. Igarashi H. Takai Y. Biochem. Biophys. Res. Commun. 1995; 206: 439-448Crossref PubMed Scopus (110) Google Scholar). Interestingly, six Arg residues are clustered at one of the putative Ca2+ binding loops in the Doc2γ C2A domain (see Fig. 3, #). Our deletion and mutation analyses indicate that these basic residues are essential for nuclear localization of Doc2γ instead of Ca2+ binding.Figure 3Alignment of the putative Ca2+binding loop 3 of the two C2 domains of the mouse C-type tandem C2 protein family. Residues, half of whose sequences were conserved or were similar, are shown on a black background andshaded background, respectively. Asterisksindicate the conserved Asp or Glu residues, which may be crucial for Ca2+ binding by analogy with the Syt I-C2A domain (44Sutton R.B. Ernst J.A. Brunger A.T. J. Cell Biol. 1999; 147: 589-598Crossref PubMed Scopus (157) Google Scholar, 48Ubach J. Zhang X. Shao X. Südhof T.C. Rizo J. EMBO J. 1998; 17: 3921-3930Crossref PubMed Scopus (250) Google Scholar). The number signs (#) indicate the basic (six Arg) residues that are only conserved in the C2A domain of Doc2γ. The location of the β-strands is indicated by arrows (44Sutton R.B. Ernst J.A. Brunger A.T. J. Cell Biol. 1999; 147: 589-598Crossref PubMed Scopus (157) Google Scholar, 48Ubach J. Zhang X. Shao X. Südhof T.C. Rizo J. EMBO J. 1998; 17: 3921-3930Crossref PubMed Scopus (250) Google Scholar). Amino acidnumbers are indicated on the right. The amino acid sequences of the mouse C-type tandem C2 proteins were from Syt I, Syt II, and rabphilin-3A (12Fukuda M. Aruga J. Niinobe M. Aimoto S. Mikoshiba K. J. Biol. Chem. 1994; 269: 29206-29211Abstract Full Text PDF PubMed Google Scholar), Syts III and IV (21Fukuda M. Kojima T. Aruga J. Niinobe M. Mikoshiba K. J. Biol. Chem. 1995; 270: 26523-26527Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar), Syts V-XI (35Fukuda M. Kanno E. Mikoshiba K. J. Biol. Chem. 1999; 274: 31421-31427Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar), Syt XIII (29Fukuda M. Mikoshiba K. Biochem. J. 2001; 354: 249-257Crossref PubMed Scopus (39) Google Scholar), granuphilin-a (46Wang J. Takeuchi T. Yokota H. Izumi T. J. Biol. Chem. 1999; 274: 28542-28548Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar), Doc2α (53Naito A. Orita S. Wanaka A. Sasaki T. Sakaguchi G. Maeda M. Igarashi H. Tohyama M. Takai Y. Mol. Brain Res. 1997; 44: 198-204Crossref PubMed Scopus (19) Google Scholar), Doc2β (32Kojima T. Fukuda M. Aruga J. Mikoshiba K. J. Biochem. ( Tokyo ). 1996; 120: 671-676Crossref PubMed Scopus (45) Google Scholar), Doc2γ (30Fukuda M. Mikoshiba K. Biochem. Biophys. Res. Commun. 2000; 276: 626-632Crossref PubMed Scopus (34) Google Scholar), Slp1–3 (45Fukuda M. Mikoshiba K. Biochem. Biophys. Res. Commun. 2001; 281: 1226-1233Crossref PubMed Scopus (80) Google Scholar), and Syt B/K and Syt XII.2View Large Image Figure ViewerDownload (PPT) pEF-T7-Doc2γ, -Doc2γΔC2AB (amino acid residues 1–80), -Doc2γΔC2B (amino acid residues 1–217), -Doc2γ-C2A (amino acid residues 80–217), Doc2γ-C2B (amino acid residues 234–388), and T7-Doc2β (amino acid residues 1–418) (32Kojima T. Fukuda M. Aruga J. Mikoshiba K. J. Biochem. ( Tokyo ). 1996; 120: 671-676Crossref PubMed Scopus (45) Google Scholar) were constructed by polymerase chain reaction (PCR) using the following sets of primers with appropriate restriction enzyme sites (underlined) and/or termination codons (bold letters), as described previously (19Fukuda M. Mikoshiba K. J. Biol. Chem. 2000; 275: 28180-28185Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 35Fukuda M. Kanno E. Mikoshiba K. J. Biol. Chem. 1999; 274: 31421-31427Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar): 5′-CGGATCCATGGCATGTGCAGGGCCAGCC-3′ (Met primer; sense), 5′-GCACTAGT CAGTCATCCGAGTCTCCTTC-3′ (ΔC2AB primer; antisense), 5′-GCGGATCCGACAGCACTGCCCTAGGCAC-3′ (C2A upper primer; sense), 5′-GCACTAGT CACCTCTTGGTCAGCTTCCGCT-3′ (C2A lower primer; antisense), 5′-GCGGATCCGAGGTGGAGGCAGAGGTGTT-3′ (C2B upper primer; sense), 5′-GCTGACTAGT CACCAAGTT-3′ (C1 primer; antisense), 5′-GCGGATCCATGACCCTCCGGCGGCGCGGGGAGAAGGCGACCATCAGCA-3′ (Doc2β-Met primer; sense), and 5′-GCACTAGT CAGTCGCTGAGTACAGC-3′ (Doc2β-stop primer; antisense). Briefly, purified PCR products digested with BamHI and SpeI were subcloned into the BamHI/SpeI site of a modified pEF-BOS vector with a T7 tag (19Fukuda M. Mikoshiba K. J. Biol. Chem. 2000; 275: 28180-28185Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 35Fukuda M. Kanno E. Mikoshiba K. J. Biol. Chem. 1999; 274: 31421-31427Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 36Mizushima S. Nagata S. Nucleic Acids Res. 1990; 18: 5332Crossref Scopus (1501) Google Scholar) and verified by DNA sequencing with a Hitachi SQ-5500 DNA sequencer. Plasmid DNA was prepared by using Wizard® minipreps (Promega, Madison, WI) or Maxi prep kits (Qiagen, Chatsworth, CA). A mutant Doc2γΔR (deletion of amino acids 180–183 (four Arg residues) in the C2A domain) was essentially produced by means of two-step PCR techniques, as described previously (21Fukuda M. Kojima T. Aruga J. Niinobe M. Mikoshiba K. J. Biol. Chem. 1995; 270: 26523-26527Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar), using the following pairs of oligonucleotides: Met primer and 5′-GGGGGGCCCCCGCAGCCGTGAGTCCTC-3′ (ΔR-5′ primer; antisense) (left half); and 5′-CGGGGGCCCCCCCTGGGGGAGCTA-3′ (ΔR-3′ primer; sense) and C1 primer (right half). Briefly, the right and left halves were separately amplified by using pGEM-T-Doc2γ (30Fukuda M. Mikoshiba K. Biochem. Biophys. Res. Commun. 2000; 276: 626-632Crossref PubMed Scopus (34) Google Scholar) as a template, and the two resulting PCR fragments were digested withApaI (underlined above), ligated to each other, and reamplified with the Met and C1 primers. The PCR fragment obtained that encoded the mutant Doc2γΔR was digested withBamHI/SpeI, inserted into theBamHI/SpeI site of the pEF-T7 tag vector (19Fukuda M. Mikoshiba K. J. Biol. Chem. 2000; 275: 28180-28185Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar,35Fukuda M. Kanno E. Mikoshiba K. J. Biol. Chem. 1999; 274: 31421-31427Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar), and verified by DNA sequencing. A mutant Doc2β(R6) was similarly constructed by using the following mutagenic oligonucleotides: 5′-GGGCCCTCGCCGCCGGCGCCGCAGCCGTGACTCATCACACACGGAGAT-3′ (Doc2β(R6)-5′ primer; antisense) and 5′-GGGCCCCCCATTGGAGAGACTCGGGTGCCC-3′ (Doc2β(R6)-3′ primer; antisense). Transfection of pEF-T7-Doc2 into PC12 cells (0.5–1 × 105 cells, the day before transfection/35-mm dish; MatTek Corp., Ashland, MA) or into COS-7 cells (5 × 105 cells, the day before transfection/10-cm dish) was performed as described previously (19Fukuda M. Mikoshiba K. J. Biol. Chem. 2000; 275: 28180-28185Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 35Fukuda M. Kanno E. Mikoshiba K. J. Biol. Chem. 1999; 274: 31421-31427Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar,37Fukuda M. Mikoshiba K. J. Biol. Chem. 1999; 274: 31428-31434Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). After washing twice with phosphate-buffered saline, the PC12 cells were fixed, incubated with anti-T7 tag mouse monoclonal antibody (1/5000 dilution; Novagen, Madison, WI) and anti-p300 rabbit polyclonal antibody (1/500 dilution; Santa Cruz Biotechnology, Santa Cruz, CA), and then visualized with anti-mouse Alexa 488 and anti-rabbit Alexa 568 antibodies (1/5000 dilution; Molecular Probes, Eugene, OR), respectively. In some cases, Vectashield mounting medium with DAPI (Vector Laboratories, Burlingame, CA) was added after immunostaining with anti-T7 tag antibody. Immunoreactivity was analyzed with a fluorescence microscope (TE300; Nikon, Tokyo, Japan) attached to a laser confocal scanner unit CSU 10 (Yokogawa Electric Corp., Tokyo, Japan) and HiSCA CCD camera (C6790; Hamamatsu Photonics, Hamamatsu, Japan). Images were pseudo-colored and superimposed with Adobe PhotoShop software (Version 4.0). GlutathioneS-transferase (GST) fusion proteins were expressed and purified on glutathione-Sepharose (Amersham Pharmacia Biotech) by the standard method (38Smith D.B. Johnson K.S. Gene. 1988; 67: 31-40Crossref PubMed Scopus (5046) Google Scholar). Preparation of liposomes consisting of l-α-phosphatidylcholine (PC), dipalmitoyl, and l-α-phosphatidylserine (PS), dioleoyl (1:1 w/w), and a phospholipid binding assay were performed as described previously (13Fukuda M. Kojima T. Mikoshiba K. J. Biol. Chem. 1996; 271: 8430-8434Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 33Fukuda M. Kojima T. Mikoshiba K. Biochem. J. 1997; 323: 421-425Crossref PubMed Scopus (39) Google Scholar). Proteins bound to the PS/PC liposomes were analyzed by 10% SDS-polyacrylamide gel electrophoresis and then stained with Coomassie Brilliant Blue R-250. The protein concentrations were determined with a Bio-Rad protein assay kit (Bio-Rad) by using bovine serum albumin for reference. The Doc2 family consists of three isoforms (α, β, and γ) in rats and mice (30Fukuda M. Mikoshiba K. Biochem. Biophys. Res. Commun. 2000; 276: 626-632Crossref PubMed Scopus (34) Google Scholar, 32Kojima T. Fukuda M. Aruga J. Mikoshiba K. J. Biochem. ( Tokyo ). 1996; 120: 671-676Crossref PubMed Scopus (45) Google Scholar, 34Orita S. Sasaki T. Naito A. Komuro R. Ohtsuka T. Maeda M. Suzuki H. Igarashi H. Takai Y. Biochem. Biophys. Res. Commun. 1995; 206: 439-448Crossref PubMed Scopus (110) Google Scholar, 39Sakaguchi G. Orita S. Maeda M. Igarashi H. Takai Y. Biochem. Biophys. Res. Commun. 1995; 217: 1053-1061Crossref PubMed Scopus (62) Google Scholar) and shares a highly conserved amino-terminal Munc13–1 interacting domain (Mid domain; amino acid residues 13–37 of Doc2α) (40Orita S. Naito A. Sakaguchi G. Maeda M. Igarashi H. Sasaki T. Takai Y. J. Biol. Chem. 1997; 272: 16081-16084Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar) and two C2 domains at the carboxyl terminus (the C2A domain and the C2B domain) (Fig. 1 A). Although this carboxyl-terminal tandem C2 domain structure is also found in the synaptotagmin family and rabphilin-3A, the Doc2 family is distinguished from other tandem C2 protein families in possessing a Mid domain at their amino terminus (6Südhof T.C. Rizo J. Neuron. 1996; 17: 379-388Abstract Full Text Full Text PDF PubMed Scopus (396) Google Scholar, 40Orita S. Naito A. Sakaguchi G. Maeda M. Igarashi H. Sasaki T. Takai Y. J. Biol. Chem. 1997; 272: 16081-16084Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). Doc2α is specifically expressed in neuronal cells (34Orita S. Sasaki T. Naito A. Komuro R. Ohtsuka T. Maeda M. Suzuki H. Igarashi H. Takai Y. Biochem. Biophys. Res. Commun. 1995; 206: 439-448Crossref PubMed Scopus (110) Google Scholar, 41Verhage M. de Vries K.J. Røshol H. Burbach J.P.H. Gispen W.H. Südhof T.C. Neuron. 1997; 18: 453-461Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar), whereas Doc2β and Doc2γ are expressed ubiquitously (30Fukuda M. Mikoshiba K. Biochem. Biophys. Res. Commun. 2000; 276: 626-632Crossref PubMed Scopus (34) Google Scholar, 32Kojima T. Fukuda M. Aruga J. Mikoshiba K. J. Biochem. ( Tokyo ). 1996; 120: 671-676Crossref PubMed Scopus (45) Google Scholar, 39Sakaguchi G. Orita S. Maeda M. Igarashi H. Takai Y. Biochem. Biophys. Res. Commun. 1995; 217: 1053-1061Crossref PubMed Scopus (62) Google Scholar). Both Doc2α and Doc2β have been shown to be associated with synaptic vesicle fractions in the brain (34Orita S. Sasaki T. Naito A. Komuro R. Ohtsuka T. Maeda M. Suzuki H. Igarashi H. Takai Y. Biochem. Biophys. Res. Commun. 1995; 206: 439-448Crossref PubMed Scopus (110) Google Scholar, 41Verhage M. de Vries K.J. Røshol H. Burbach J.P.H. Gispen W.H. Südhof T.C. Neuron. 1997; 18: 453-461Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar), but the subcellular localization of Doc2γ has yet to be determined. To address this we expressed T7-tagged Doc2γ proteins in PC12 cells. To our surprise the Doc2γ proteins were almost exclusively localized in the nucleus and overlapped well with p300 transcription factor and DAPI (Fig. 1 B, top panels, and data not shown). The Doc2γ proteins seemed to be uniformly present throughout the nucleoplasm. By contrast, Doc2β proteins are mainly present in the cytosol, the same as Doc2α proteins (Fig. 1 B, bottom panels) (42Orita S. Sasaki T. Komuro R. Sakaguchi G. Maeda M. Igarashi H. Takai Y. J. Biol. Chem. 1996; 271: 7257-7260Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). To determine which domain is essential for the nuclear localization of Doc2γ, we produced four deletion mutants, each of which involves a different domain of Doc2γ (Doc2γΔC2AB, Doc2γΔC2B, Doc2γ-C2A, and Doc2γ-C2B; see Fig.2 A). First, we checked the size of the mutants by immunoblotting and confirmed that they were expressed correctly, with no degradation (Fig. 2 B). Each deletion mutant was then expressed in PC12 cells, and its subcellular localization was determined by immunocytochemistry, as described above (Fig. 2 C). Interestingly, both the Doc2γΔC2B and Doc2γ-C2A proteins showed nuclear localization in PC12 cells, whereas the amino-terminal Mid domain was localized in the cytosol, and the Doc2γ-C2B protein was localized in both the nucleus and the cytosol. We therefore concluded that only the C2A domain contains a functional nuclear localization signal. Various nuclear localization signals have been determined in many proteins localized in nucleus, and they have often consisted of clusters of basic residues (Arg and Lys; reviewed in Ref. 43Jans D.A. Xiao C.-Y. Lam M.H.C. Bioessays. 2000; 22: 532-544Crossref PubMed Scopus (477) Google Scholar). Consistent with this, we found that the Doc2γ-C2A domain contains a cluster of basic residues (177RLRRRRR183) in the putative Ca2+ binding loop 3, between the β6 and β7 strands (Fig. 3, #) (44Sutton R.B. Ernst J.A. Brunger A.T. J. Cell Biol. 1999; 147: 589-598Crossref PubMed Scopus (157) Google Scholar). Interestingly, the loop 3 domain of the Doc2γ C2A domain is three amino acids longer than in other carboxyl-terminal type (C-type) tandem C2 protein families, including Syts I-XIII (9Marquèze B. Berton F. Seagar M. Biochimie ( Paris ). 2000; 82: 409-420Crossref PubMed Scopus (90) Google Scholar, 29Fukuda M. Mikoshiba K. Biochem. J. 2001; 354: 249-257Crossref PubMed Scopus (39) Google Scholar), Slp1–3 (synaptotagmin-like protein) (45Fukuda M. Mikoshiba K. Biochem. Biophys. Res. Commun. 2001; 281: 1226-1233Crossref PubMed Scopus (80) Google Scholar), granuphilin-a (46Wang J. Takeuchi T. Yokota H. Izumi T. J. Biol. Chem. 1999; 274: 28542-28548Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar), rabphilin-3A (47Shirataki H. Kaibuchi K. Sakoda T. Kishida S. Yamaguchi T. Wada K. Miyazaki M. Takai Y. Mol. Cell. Biol. 1993; 13: 2061-2068Crossref PubMed Scopus (355) Google Scholar), and other members of the Doc2 family (31Duncan R.R. Shipston M.J. Chow R.H. Biochimie ( Paris ). 2000; 82: 421-426Crossref PubMed Scopus (41) Google Scholar). It is also noteworthy that other C-type tandem C2 domains do not contain an Arg cluster at this position (Fig. 3). Consistent with this, there have been no reports of tandem C2 proteins that specifically localized in nucleus. Although three Asp residues between the β6 and β7 strands in the C2A domain of Syt I (asterisks in Fig. 3) are known to bind Ca2+ions (48Ubach J. Zhang X. Shao X. Südhof T.C. Rizo J. EMBO J. 1998; 17: 3921-3930Crossref PubMed Scopus (250) Google Scholar), the C2A domain of Doc2γ lacks two Asp residues (Ser-176 and Pro-185), and because of these amino acid substitutions, the Doc2γ C2A domain does not display any clear Ca2+-dependent phospholipid (PS/PC liposome) binding activity (Fig. 4 B) (30Fukuda M. Mikoshiba K. Biochem. Biophys. Res. Commun. 2000; 276: 626-632Crossref PubMed Scopus (34) Google Scholar). To determine whether the basic cluster of the Doc2γ C2A domain is the sole nuclear localization signal of this protein, we produced a deletion mutant lacking four of six Arg residues (named Doc2γΔR; see Fig. 4 A). As expected, the Doc2γΔR proteins were mainly localized in the cytosol of PC12 cells and mostly absent in the nucleus (Fig. 4 C, top panels). Finally, we investigated whether the basic cluster alone of Doc2γ is a sufficient nuclear localization signal by producing chimera proteins between Doc2β and Doc2γ in which the loop 3 domain of Doc2β was replaced by that of Doc2γ (named Doc2β(R6); see Fig. 4 A). As a result of this substitution, the Doc2β(R6) C2A domain completely lost its Ca2+-dependent phospholipid binding activity (Fig. 4 B), whereas the Doc2β(R6) proteins acquired the ability to localize in the nucleus of PC12 cells (Fig.4 C, bottom panels). These findings indicate that the basic cluster of Doc2γ is both necessary and sufficient for nuclear localization of Doc2γ protein. This study revealed the novel function of the Ca2+-independent type of the Doc2γ C2A domain in nuclear localization. It is noteworthy that the basic cluster (RLRRRRR) is present in the putative Ca2+ binding loop 3, which is located at the apex of β-sandwich structure of the Doc2γ C2A domain (i.e. loop 3 functions as a nuclear localization signal rather than a Ca2+-binding site). Thus, the function of the loop domains of the C2 domain is more diversified than we expected. The function of Doc2γ in the nucleus remains unclear, but because Doc2α isoform is involved in secretory vesicle exocytosis (42Orita S. Sasaki T. Komuro R. Sakaguchi G. Maeda M. Igarashi H. Takai Y. J. Biol. Chem. 1996; 271: 7257-7260Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 49Mochida S. Orita S. Sakaguchi G. Sasaki T. Takai Y. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11418-11422Crossref PubMed Scopus (69) Google Scholar, 50Sakaguchi G. Manabe T. Kobayashi K. Orita S. Sasaki T. Naito A. Maeda M. Igarashi H. Katsuura G. Nishioka H. Mizoguchi A. Itohara S. Takahashi T. Takai Y. Eur. J. Neurosci. 1999; 11: 4262-4268Crossref PubMed Scopus (50) Google Scholar), and vesicle traffic is thought to be regulated by a conserved protein family, such as SNARE (solubleN -ethylmaleimide-sensitive factorattachment protein receptor) proteins, C-type tandem C2 protein families, and rab family (51Jahn R. Südhof T.C. Annu. Rev. Biochem. 1999; 68: 863-911Crossref PubMed Scopus (1021) Google Scholar, 52Lin R.C. Scheller R.H. Annu. Rev. Cell Dev. Biol. 2000; 16: 19-49Crossref PubMed Scopus (422) Google Scholar), Doc2γ might be involved in nuclear envelope assembly. As far as we know, Doc2γ is the only isoform of the C-type tandem C2 protein family that is localized in the nucleus. Further work is necessary to elucidate whether Doc2γ regulates nuclear envelope assembly.
C2 domain
HAMP domain
Protein kinase domain
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Human papillomavirus (HPV) entry is accompanied by multiple receptor-induced conformational changes (CCs) affecting both the major and minor capsid proteins, L1 and L2. Interaction of heparan sulfate (HS) with L1 is essential for successful HPV16 entry. Recently, cocrystallization of HPV16 with heparin revealed four distinct binding sites. Here we characterize mutant HPV16 to delineate the role of engagement with HS binding sites during infectious internalization. Site 1 (Lys278, Lys361), which mediates primary binding, is sufficient to trigger an L2 CC, exposing the amino terminus. Site 2 (Lys54, Lys356) and site 3 (Asn57, Lys59, Lys442, Lys443) are engaged following primary attachment and are required for infectious entry. Site 2 mutant particles are efficiently internalized but fail to undergo an L1 CC on the cell surface and subsequent uncoating in the endocytic compartment. After initial attachment to the cell, site 3 mutants undergo L1 and L2 CCs and then accumulate on the extracellular matrix (ECM). We conclude that the induction of CCs following site 1 and site 2 interactions results in reduced affinity for the primary HS binding site(s) on the cell surface, which allows engagement with site 3. Taken together, our findings suggest that HS binding site engagement induces CCs that prepare the virus for downstream events, such as the exposure of secondary binding sites, CCs, transfer to the uptake receptor, and uncoating.
Internalization
A-site
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Serum Albumin
Human serum albumin
Bovine serum albumin
A-site
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