The Reovirus σ1 Aspartic Acid Sandwich

Reovirus attachment protein σ1 mediates engagement of receptors on the surface of target cells and undergoes dramatic conformational rearrangements during viral disassembly in the endocytic pathway. The σ1 protein is a filamentous, trimeric molecule with a globular β-barrel head domain. An unusual cluster of aspartic acid residues sandwiched between hydrophobic tyrosines is located at the σ1 subunit interface. A 1.75-Å structure of the σ1 head domain now reveals two water molecules at the subunit interface that are held strictly in position and interact with neighboring residues. Structural and biochemical analyses of mutants affecting the aspartic acid sandwich indicate that these residues and the corresponding chelated water molecules act as a plug to block the free flow of solvent and stabilize the trimer. This arrangement of residues at the σ1 head trimer interface illustrates a new protein design motif that may confer conformational mobility during cell entry. Reovirus attachment protein σ1 mediates engagement of receptors on the surface of target cells and undergoes dramatic conformational rearrangements during viral disassembly in the endocytic pathway. The σ1 protein is a filamentous, trimeric molecule with a globular β-barrel head domain. An unusual cluster of aspartic acid residues sandwiched between hydrophobic tyrosines is located at the σ1 subunit interface. A 1.75-Å structure of the σ1 head domain now reveals two water molecules at the subunit interface that are held strictly in position and interact with neighboring residues. Structural and biochemical analyses of mutants affecting the aspartic acid sandwich indicate that these residues and the corresponding chelated water molecules act as a plug to block the free flow of solvent and stabilize the trimer. This arrangement of residues at the σ1 head trimer interface illustrates a new protein design motif that may confer conformational mobility during cell entry. Mammalian orthoreoviruses (reoviruses) 5The abbreviations used are: reoviruses, mammalian orthoreoviruses; JAM-A, junctional adhesion molecule-A; T3D, type 3 Dearing; GST, glutathione S-transferase; VSV, vesicular stomatitis virus. attach to cells by specific binding to both carbohydrate and proteinaceous receptors. For serotype 3 reoviruses, viral attachment is a multistep process initiated by low affinity binding to sialic acid followed by high affinity binding to junctional adhesion molecule-A (JAM-A) (1Barton E.S. Connolly J.L. Forrest J.C. Chappell J.D. Dermody T.S. J. Biol. Chem. 2001; 276: 2200-2211Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 2Barton E.S. Forrest J.C. Connolly J.L. Chappell J.D. Liu Y. Schnell F. Nusrat A. Parkos C.A. Dermody T.S. Cell. 2001; 104: 441-451Abstract Full Text Full Text PDF PubMed Scopus (519) Google Scholar). These steps are mediated by discrete receptor-binding domains in the attachment protein, σ1 (3Chappell J.D. Duong J.L. Wright B.W. Dermody T.S. J. Virol. 2000; 74: 8472-8479Crossref PubMed Scopus (104) Google Scholar), a fiber-like molecule with head-and-tail morphology (4Furlong D.B. Nibert M.L. Fields B.N. J. Virol. 1988; 62: 246-256Crossref PubMed Google Scholar, 5Fraser R.D.B. Furlong D.B. Trus B.L. Nibert M.L. Fields B.N. Steven A.C. J. Virol. 1990; 64: 2990-3000Crossref PubMed Google Scholar, 6Chappell J.D. Prota A. Dermody T.S. Stehle T. EMBO J. 2002; 21: 1-11Crossref PubMed Scopus (174) Google Scholar). Strain type 3 Dearing (T3D) σ1 has distinct binding sites for its receptors: the head domain binds to JAM-A with high affinity (2Barton E.S. Forrest J.C. Connolly J.L. Chappell J.D. Liu Y. Schnell F. Nusrat A. Parkos C.A. Dermody T.S. Cell. 2001; 104: 441-451Abstract Full Text Full Text PDF PubMed Scopus (519) Google Scholar), whereas a region in the tail has been implicated in binding to sialic acid (1Barton E.S. Connolly J.L. Forrest J.C. Chappell J.D. Dermody T.S. J. Biol. Chem. 2001; 276: 2200-2211Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 3Chappell J.D. Duong J.L. Wright B.W. Dermody T.S. J. Virol. 2000; 74: 8472-8479Crossref PubMed Scopus (104) Google Scholar, 7Chappell J.D. Gunn V.L. Wetzel J.D. Baer G.S. Dermody T.S. J. Virol. 1997; 71: 1834-1841Crossref PubMed Google Scholar). Viral attachment to the cell surface by σ1 leads to internalization of the virus by receptor-mediated endocytosis that is likely clathrin-dependent (8Sturzenbecker L.J. Nibert M.L. Furlong D.B. Fields B.N. J. Virol. 1987; 61: 2351-2361Crossref PubMed Google Scholar, 9Baer G.S. Ebert D.H. Chung C.J. Erickson A.H. Dermody T.S. J. Virol. 1999; 73: 9532-9543Crossref PubMed Google Scholar, 10Ehrlich M. Boll W. Van Oijen A. Hariharan R. Chandran K. Nibert M.L. Kirchhausen T. Cell. 2004; 118: 591-605Abstract Full Text Full Text PDF PubMed Scopus (691) Google Scholar). Within endosomes, virions undergo acid-dependent, proteolytic disassembly to form infectious subvirion particles (8Sturzenbecker L.J. Nibert M.L. Furlong D.B. Fields B.N. J. Virol. 1987; 61: 2351-2361Crossref PubMed Google Scholar, 11Baer G.S. Dermody T.S. J. Virol. 1997; 71: 4921-4928Crossref PubMed Google Scholar). Infectious subvirion particles penetrate endosomal membranes and release transcriptionally active cores into the cytoplasm (12Chandran K. Farsetta D.L. Nibert M.L. J. Virol. 2002; 76: 9920-9933Crossref PubMed Scopus (142) Google Scholar, 13Chandran K. Parker J.S. Ehrlich M. Kirchhausen T. Nibert M.L. J. Virol. 2003; 77: 13361-13375Crossref PubMed Scopus (79) Google Scholar, 14Odegard A.L. Chandran K. Zhang X. Parker J.S. Baker T.S. Nibert M.L. J. Virol. 2004; 78: 8732-8745Crossref PubMed Scopus (100) Google Scholar). Accumulating evidence suggests that σ1 undergoes dramatic conformational changes during viral disassembly and that these changes facilitate key steps in the cell entry process (4Furlong D.B. Nibert M.L. Fields B.N. J. Virol. 1988; 62: 246-256Crossref PubMed Google Scholar, 15Dryden K.A. Wang G. Yeager M. Nibert M.L. Coombs K.M. Furlong D.B. Fields B.N. Baker T.S. J. Cell Biol. 1993; 122: 1023-1041Crossref PubMed Scopus (293) Google Scholar, 16Nibert M.L. Chappell J.D. Dermody T.S. J. Virol. 1995; 69: 5057-5067Crossref PubMed Google Scholar). We previously determined the structure of the C-terminal half of σ1, which comprises the head domain plus a short region of the tail (6Chappell J.D. Prota A. Dermody T.S. Stehle T. EMBO J. 2002; 21: 1-11Crossref PubMed Scopus (174) Google Scholar). This structure provided clues about the interaction of the head domain with its receptor JAM-A and the nature of trimer contacts. The head domain contains a water-filled cavity formed by three eight-stranded β-barrels, one donated by each monomer. The tail region consists of three β-spiral repeats. One of the most remarkable features of the σ1 structure is a cluster of aspartic acid residues at the base of the head domain (6Chappell J.D. Prota A. Dermody T.S. Stehle T. EMBO J. 2002; 21: 1-11Crossref PubMed Scopus (174) Google Scholar). Molecular dynamics studies suggest that these residues are likely to play a role in mediating conformational changes in σ1 (17Cavalli A. Prota A.E. Stehle T. Dermody T.S. Recanatini M. Folkers G. Scapozza L. Biophys. J. 2004; 86: 3423-3431Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar). However, at 2.6 Å resolution, the structure did not allow precise placement of water molecules and visualization of contacts between amino acids with sufficient accuracy to explain how such a unique arrangement of amino acids is compatible with a higher order structure. In this study, we determined a high resolution structure of a fragment of T3D σ1 that comprises the head domain and a single β-spiral repeat of the tail. The structure has been refined to a resolution of 1.75 Å, allowing us to discern with high clarity details of the subunit interface in the vicinity of the aspartic acid cluster. Furthermore, we have analyzed two σ1 mutants with alterations in the vicinity of the subunit interface to determine the effects on receptor binding capacity and trimer stability. Our studies suggest that the aspartic acid cluster serves as a molecular switch that, depending on the microenvironment, can stabilize or destabilize the formation of a trimeric structure. Protein Expression, Purification, and Analysis—A cDNA encoding residues 293-455 of T3D σ1 was amplified by PCR and introduced into pGEX4T-3 (GE Healthcare). Mutations were engineered using site-directed PCR with appropriate mutagenic primers. Expression was induced with 0.2 mm IPTG in Escherichia coli strain BL21(DE3) pLys-S cells (Novagen) at 25 °C. Bacteria were centrifuged to form a pellet, solubilized in 50 mm Tris (pH 7.8), 3 mm EDTA, 1% Triton X-100, 2 mm β-mercaptoethanol, 1 mm phenylmethylsulfonyl fluoride, and 100 μg/ml lysozyme, submitted to 50% duty-cycle sonication pulses using a Branson Sonifier 450, and centrifuged at 15,000 × g. The soluble fraction was purified using a 5-ml GSTrapFF column (GE Healthcare) and eluted with 30 mm reduced glutathione, 2 mm β-mercaptoethanol, 3 mm EDTA, and 50 mm Tris (pH 8.05). Controlled tryptic protease treatment was performed overnight at 4 °C to remove the glutathione S-transferase (GST) tag. The sample was equilibrated using PD-10 desalting columns and purified further using Mono Q anion-exchange chromatography (GE Healthcare) with an increasing gradient of NaCl in 20 mm HEPES (pH 7.1). The σ1-Y313A mutant protein was cleaved with thrombin on-column and further purified by gel filtration using a Superdex 75 column (GE Healthcare). Human JAM-A (hJAM-A), expressed as a GST fusion protein, was purified as described (18Prota A.E. Campbell J.A. Schelling P. Forrest J.C. Peters T.R. Watson M.J. Aurrand-Lions M. Imhof B. Dermody T.S. Stehle T. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 5366-5371Crossref PubMed Scopus (134) Google Scholar). Analytical scale gel filtration was performed using a Superdex 75 column mounted on a SMART system (GE Healthcare) in 20 mm Tris (pH 7.5), 100 mm NaCl. Protein Crystallization and Data Collection—Purified T3D σ1 head domain was subjected to size-exclusion chromatography in 20 mm Tris (pH 7.5), 100 mm NaCl, and 0.01% sodium azide and concentrated using Millipore 5 MWCO filters to 13.6 mg/ml, as assessed by direct measurement of absorbance at 260 and 280 nm using the relationship: c[mg/ml] = (1.55 × A280 nm) - (0.76 × A260 nm), since the T3D σ1 head domain does not react linearly with either Bradford or Lowry dyes (data not shown). Crystals of native σ1 protein were grown from 10-12% polyethylene glycol 8000, 0.2 m magnesium sulfate, and 0.1 m sodium cacodylate (pH 6.9) by mixing an equal amount of protein and precipitant solution. was concentrated to mg/ml, and were grown using the used to of the protein with polyethylene glycol Crystals were using as Data from were at the features high and Data for were at the of the using a were from single and with W. 1997; 276: PubMed Scopus Google Scholar). of σ1 to = Å, = Å, = Å, = and two in the The structure was determined by molecular using the trimeric σ1 head domain (6Chappell J.D. Prota A. Dermody T.S. Stehle T. EMBO J. 2002; 21: 1-11Crossref PubMed Scopus (174) Google as a in J. A. Scopus Google Scholar). of and were performed using the M. A. PubMed Scopus Google and P. J.S. J. M. T. Biol. PubMed Scopus Google Scholar), The P. K. Biol. 2004; PubMed Scopus Google and Biol. PubMed Scopus Google were used to the formed that to with cell The structure of this protein was and refined to Å using and and have been with the Protein Data with and for the and mutant and for the σ1 and were at 100 and a of Å and Å in to the resolution Å for σ1 and Å for cell = = = = = = = = = - is the of a and is the of = - and are and structure contains of the free = - and are and structure contains of the of water with Data were at 100 and a of Å and Å in to the resolution Å for σ1 and Å for = - is the of a and is the of = - and are and structure PubMed Scopus Google contains of the with Biol. PubMed Scopus Google Scholar). in a new was with to protein by GST or fusion protein at a of 2 in (pH were by flow cells of an for 3 at 30 using a T3D σ1 head domain was the surface at 30 σ1 were with a of mm (pH for σ1 binding to were determined using and with a binding R. A. J. 1997; PubMed Scopus Google Scholar). of the σ1 previously T3D σ1 protein (6Chappell J.D. Prota A. Dermody T.S. Stehle T. EMBO J. 2002; 21: 1-11Crossref PubMed Scopus (174) Google a between the C-terminal two β-spiral of the tail. This likely to the of 2.6 Å for these of T3D σ1, we a the head domain and the C-terminal β-spiral repeat of the tail We an the head domain did not soluble and trimeric protein, that the C-terminal β-spiral to trimer The protein was with a GST and purified glutathione affinity chromatography and gel filtration that the protein at an molecular of with a trimer that the protease treatment was the σ1 head domain with two amino acids from the protease (data not shown). of the σ1 to determine the purified protein we assessed the capacity of the σ1 head domain to to JAM-A using surface The JAM-A expressed and purified as a GST fusion protein (18Prota A.E. Campbell J.A. Schelling P. Forrest J.C. Peters T.R. Watson M.J. Aurrand-Lions M. Imhof B. Dermody T.S. Stehle T. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 5366-5371Crossref PubMed Scopus (134) Google Scholar), was on a surface with a the purified σ1 head and to the not to GST of the interaction using a of × a that the determined for a fragment of σ1 (2Barton E.S. Forrest J.C. Connolly J.L. Chappell J.D. Liu Y. Schnell F. Nusrat A. Parkos C.A. Dermody T.S. Cell. 2001; 104: 441-451Abstract Full Text Full Text PDF PubMed Scopus (519) Google Scholar). These indicate that the purified σ1 head domain is and that the domain of σ1 residues of the σ1 at 1.75 Å were at the using of the purified σ1 head domain. The structure was by molecular using the previously determined resolution structure of a C-terminal fragment of σ1 (6Chappell J.D. Prota A. Dermody T.S. Stehle T. EMBO J. 2002; 21: 1-11Crossref PubMed Scopus (174) Google and refined to 1.75 Å An for a of the refined structure that the is both and free PubMed Scopus Google are low that the structure is refined and of high A of into two are in the of the A of one trimer is in σ1 is of an eight-stranded The of the is with the of two and the at the of each and the at its the of σ1 residues in the Biol. PubMed Scopus Google Scholar). However, since the corresponding to is that a to its in its is located in the region of the with a between and The this region as a type B.L. PubMed Scopus Google Scholar). in the σ1 formation from solvent an of monomer. in the tail for about of this hydrophobic between the of the The three β-barrel domains that form the head in a of contacts between the at the of each in binding that from the base of the head to its However, a cavity at the of the and this cavity is by that contacts The cavity Å in and Å in and contains a of water molecules that are to the surface at the of the trimer The cavity contains water which are not in of the the cavity suggest that water molecules can flow to the of the trimer. In the of the cavity is by the three The unusual cluster of aspartic acid residues the water-filled two aspartic acid and to this to a of aspartic acid that are in and are located at the of a between and A and are by a F. M. L. Biol. PubMed Scopus Google Scholar). The of the three each with the of key contacts. The aspartic acids are between two that each three at the and at the and a with from a neighboring the are not in residues or with that The of three in such a hydrophobic in at the high resolution the and of the suggests that are A of is by molecular dynamics studies of σ1 (17Cavalli A. Prota A.E. Stehle T. Dermody T.S. Recanatini M. Folkers G. Scapozza L. Biophys. J. 2004; 86: 3423-3431Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar). two One of these the of the and the of a neighboring a is formed between the and the of in a neighboring monomer. these two three and since are in head the aspartic acid sandwich likely a to trimer stability. The cluster of aspartic acids is sandwiched between hydrophobic residues that block of solvent molecules to the One of this sandwich in is formed by and the in is formed by and water molecules located on the are in this arrangement of amino These water molecules interact with and and are held in by an water molecules can However, residues in the vicinity of the two water molecules have that are the of the structure that mobility is low in this is that the water molecules can and of σ1 conformational changes of σ1, we of two key residues of the and The mutant were analyzed for receptor binding and the capacity to form We that of with a amino acid allow of water molecules to the aspartic acid the trimer to undergo changes by The σ1-Y313A mutant protein was expressed as a GST fusion in purified by glutathione cleaved from the GST and further purified using gel σ1-Y313A is soluble and as a by gel filtration chromatography of σ1-Y313A with each of three of to to the mobility of the mutant protein (data not shown). σ1-Y313A was of binding to JAM-A by either gel filtration (data not or surface that a trimeric form of σ1 is for JAM-A of σ1-Y313A a structure to that of σ1, that σ1-Y313A is (data not shown). indicate that is for and that the trimeric form of σ1 is for receptor and of σ1 determine and changes of with we engineered a in the T3D σ1 head We that form between the the trimer interface in the as by the The mutant protein was purified using the for of the T3D σ1 head domain. The mutant at and binds to JAM-A with an affinity to that of σ1 is and from the the in the T3D σ1 head domain the of the aspartic acid we determined the structure of The mutant protein was using to to the We a from the to Å and the structure by molecular using the protein structure as a The mutant as a trimer with a structure that is to that of the T3D σ1 head domain the subunit the two water molecules and residues are at the position in The amino of form the as the of the mutant structure evidence that in σ1 residues of σ1 a C-terminal fragment of σ1 has been (6Chappell J.D. Prota A. Dermody T.S. Stehle T. EMBO J. 2002; 21: 1-11Crossref PubMed Scopus (174) Google Scholar), further at an of resolution has an of its trimeric that form the trimer are The base of the trimer is held by hydrophobic a of the formation of In contacts at the and of the trimer with water water hydrophobic and that form These of are unusual for contacts. We that the σ1 head is to as both and trimeric In the of one in protein that is A single at the base of the trimer to trimer that the affinity contacts at the and of the σ1 head are not sufficient for of the trimer. of amino acid of σ1 from and reveals that and are (6Chappell J.D. Prota A. Dermody T.S. Stehle T. EMBO J. 2002; 21: 1-11Crossref PubMed Scopus (174) Google Scholar, J.A. P. Wetzel J.D. Forrest J.C. Aurrand-Lions M. Imhof B. Stehle T. Dermody T.S. J. Virol. PubMed Scopus Google Scholar). hydrophobic residues that form the and of the aspartic acid sandwich a high of are at and in a σ1 For strain type 1 contains and and strain type 2 contains and residues at corresponding to and in T3D Furthermore, a hydrophobic or is at position and a is at position The of at these suggests that the aspartic acid sandwich serves an in of that the σ1-Y313A mutant is soluble and Furthermore, since its is to that of σ1, σ1-Y313A to However, the mutant protein does not to The most likely of these is that a trimeric form of σ1 is for engagement of We two for these the JAM-A binding may one monomer. the surface structure of the region may in the of a trimer. We that JAM-A a direct with or with residues in is not to solvent in the trimeric protein and to JAM-A at its position in the head trimer The structure of σ1 is to that of the attachment protein, (6Chappell J.D. Prota A. Dermody T.S. Stehle T. EMBO J. 2002; 21: 1-11Crossref PubMed Scopus (174) Google Scholar). the receptors for and JAM-A and and and and may viral in a T. Dermody T.S. Viral 2004; PubMed Scopus Google Scholar). We that the binds to its receptor and receptor contacts between two and a single receptor this of binding is in σ1, by alterations at the trimer interface. We a single magnesium to residues and at the of the surface of the σ1 head (data not shown). This surface of σ1 the JAM-A interface and has been to in with JAM-A T. Dermody T.S. Viral 2004; PubMed Scopus Google Scholar, J.C. Campbell J.A. Schelling P. Stehle T. Dermody T.S. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). The of and are to solvent and in to of and These residues to in with residues in the In of this of mutants of JAM-A suggests that and residues in the JAM-A interface are for with M. and T. S. A of at the structure of σ1 reveals a unique cluster of aspartic acid residues at the head trimer interface. A key of these residues is that are located at the of a by its the is and of evidence the that the of to allow formation of the trimer. molecular dynamics studies that the of at the the of the three at the base of the σ1 head (17Cavalli A. Prota A.E. Stehle T. Dermody T.S. Recanatini M. Folkers G. Scapozza L. Biophys. J. 2004; 86: 3423-3431Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar). the mutant protein into a trimeric structure that is from that of the the arrangement of water molecules in the vicinity of in both the and mutant is the is an for a aspartic acid not for a the between the two that the protein contains residues. of a that from in is likely that of to its at We performed using purified σ1 head domain of and high (data not shown). The σ1 head eluted as a trimer These evidence that the aspartic acids are and to solvent in the trimer. The that in σ1 is is by the of a cluster of aspartic acids in the protein of vesicular stomatitis virus S. S. Y. PubMed Scopus Google Scholar). the of from σ1, a trimer that features three aspartic acids that each at the trimer interface. The and of the aspartic acid in and σ1 are in In both are to between aspartic acid These in a that is to the trimer and to of the trimer. In both the aspartic acids from with low mobility in and a in both water molecules that form with the are the of the aspartic acid cluster for σ1 in viral attachment and cell and cells the endosomal and a low during the entry The structure has been as a of the molecule S. S. Y. PubMed Scopus Google Scholar). In with this is that the σ1 structure a form of the protein at low and σ1 were at the used for are from in both and may an that aspartic the aspartic acid cluster may act as a molecular switch that the protein a low that to the aspartic acids from may in that changes of viral in to binding or to are PubMed Scopus Google Scholar, R. J. J. PubMed Scopus Google Scholar, B. PubMed Scopus Google Scholar). These changes allow to previously for binding and For the virus undergoes a to This conformational is by proteolytic and leads to the formation of that a hydrophobic fusion the structure at a form of the protein, as the low is at J. W. L.J. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar). The of at the σ1 trimer interface suggests that the aspartic acid cluster serves a to a between a form of the protein that is at in the head region and a molecule in the structure on that the protein is the in the of the such a conformational facilitate during entry to viral such as internalization into the endocytic and proteolytic disassembly to form infectious subvirion We the of for this with