Crystal structure of invasin: A bacterial integrin-binding protein

Science

Volumn 286, Issue 5438, 1999, Pages 291-295

  Hamburger, Zsuzsa A ^a   Brown, Michele S ^b   Isberg, Ralph R ^b   Bjorkman, Pamela J ^a  

^a CALIFORNIA INSTITUTE OF TECHNOLOGY   (United States)

^b TUFTS UNIVERSITY SCHOOL OF MEDICINE   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

BACTERIAL PROTEIN; FIBRONECTIN; INVASIN;

ARTICLE; BINDING AFFINITY; CRYSTAL STRUCTURE; NONHUMAN; PRIORITY JOURNAL; PROTEIN ANALYSIS; PROTEIN BINDING; PROTEIN STRUCTURE; STRUCTURE ANALYSIS; YERSINIA PSEUDOTUBERCULOSIS;

ADHESINS, BACTERIAL; AMINO ACID SEQUENCE; BACTERIAL PROTEINS; BINDING SITES; CRYSTALLOGRAPHY, X-RAY; EVOLUTION, MOLECULAR; FIBRONECTINS; HYDROGEN BONDING; INTEGRINS; LIGANDS; MODELS, MOLECULAR; PROTEIN BINDING; PROTEIN CONFORMATION; PROTEIN FOLDING; PROTEIN STRUCTURE, SECONDARY; YERSINIA PSEUDOTUBERCULOSIS;

BACTERIA (MICROORGANISMS); YERSINIA; YERSINIA PSEUDOTUBERCULOSIS;

EID: 0033536633     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.286.5438.291     Document Type: Article

References (70)

1. J. C. Pepe and V. L. Miller, Infect. Agents Dis. 2, 236 (1993); A. Marra and R. R. Isberg, Infect. Immun. 65, 3412 (1997); I. B. Autenrieth and R. Firsching, J. Med. Microbiol. 44, 285 (1996).
2. J. C. Pepe and V. L. Miller, Infect. Agents Dis. 2, 236 (1993); A. Marra and R. R. Isberg, Infect. Immun. 65, 3412 (1997); I. B. Autenrieth and R. Firsching, J. Med. Microbiol. 44, 285 (1996).
3. J. C. Pepe and V. L. Miller, Infect. Agents Dis. 2, 236 (1993); A. Marra and R. R. Isberg, Infect. Immun. 65, 3412 (1997); I. B. Autenrieth and R. Firsching, J. Med. Microbiol. 44, 285 (1996).
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6. R. R. Isberg and J. M. Leong, ibid. 60, 861 (1990); R. R. Isberg and G. Tran Van Nhieu, Trends Microbiol, 2, 10 (1994).
7. R. O. Hynes, Cell 69, 11 (1992).
8. G. Tran Van Nhieu, E. S. Krukonis, A. A. Reszka, A. F. Horwitz, R. R. Isberg, J Biol. Chem. 271, 7665 (1996); G. Tran Van Nhieu and R. R. Isberg, EMBO J. 12, 1887 (1993). H. Liu, L. Magoun, J. M. Leong, Infect. Immun. 67, 2045 (1999); B. Kenny et al., Cell 91, 511 (1997).
9. G. Tran Van Nhieu, E. S. Krukonis, A. A. Reszka, A. F. Horwitz, R. R. Isberg, J Biol. Chem. 271, 7665 (1996); G. Tran Van Nhieu and R. R. Isberg, EMBO J. 12, 1887 (1993). H. Liu, L. Magoun, J. M. Leong, Infect. Immun. 67, 2045 (1999); B. Kenny et al., Cell 91, 511 (1997).
10. G. Tran Van Nhieu, E. S. Krukonis, A. A. Reszka, A. F. Horwitz, R. R. Isberg, J Biol. Chem. 271, 7665 (1996); G. Tran Van Nhieu and R. R. Isberg, EMBO J. 12, 1887 (1993). H. Liu, L. Magoun, J. M. Leong, Infect. Immun. 67, 2045 (1999); B. Kenny et al., Cell 91, 511 (1997).
11. G. Tran Van Nhieu, E. S. Krukonis, A. A. Reszka, A. F. Horwitz, R. R. Isberg, J Biol. Chem. 271, 7665 (1996); G. Tran Van Nhieu and R. R. Isberg, EMBO J. 12, 1887 (1993). H. Liu, L. Magoun, J. M. Leong, Infect. Immun. 67, 2045 (1999); B. Kenny et al., Cell 91, 511 (1997).
12. J. M. Leong, R. S. Foumier, R. R. Isberg, EMBO J. 9, 1979 (1990); M. J. Worley, I. Stojiljkovic, F. Heffron, Mol. Microbiol. 29, 1471 (1998). Using a neural network algorithm that predicts the membrane topology of integral outer membrane proteins [K. Diederichs, J. Freigang, S. Umhau, K. Zeth, J. Breed, Protein Sci. 7, 2413 (1998)], we predict that residues 142 to 494 contain 22 β strands. Thus, the structure of this portion of invasin may resemble those of the membrane-spanning portions of porins (16 to 18 β strands) [reviewed by B. K. Jap and P. J. Walian, Physiol. Rev. 76, 1073 (1996)] or FhuA (22 β strands) [K. P. Locher et al., Cell 95, 771 (1998); A. D. Ferguson, E. Hofmann, J. W. Coulton, K. Diederichs, W. Weite, Science 282, 2215 (1998)].
13. J. M. Leong, R. S. Foumier, R. R. Isberg, EMBO J. 9, 1979 (1990); M. J. Worley, I. Stojiljkovic, F. Heffron, Mol. Microbiol. 29, 1471 (1998). Using a neural network algorithm that predicts the membrane topology of integral outer membrane proteins [K. Diederichs, J. Freigang, S. Umhau, K. Zeth, J. Breed, Protein Sci. 7, 2413 (1998)], we predict that residues 142 to 494 contain 22 β strands. Thus, the structure of this portion of invasin may resemble those of the membrane-spanning portions of porins (16 to 18 β strands) [reviewed by B. K. Jap and P. J. Walian, Physiol. Rev. 76, 1073 (1996)] or FhuA (22 β strands) [K. P. Locher et al., Cell 95, 771 (1998); A. D. Ferguson, E. Hofmann, J. W. Coulton, K. Diederichs, W. Weite, Science 282, 2215 (1998)].
14. J. M. Leong, R. S. Foumier, R. R. Isberg, EMBO J. 9, 1979 (1990); M. J. Worley, I. Stojiljkovic, F. Heffron, Mol. Microbiol. 29, 1471 (1998). Using a neural network algorithm that predicts the membrane topology of integral outer membrane proteins [K. Diederichs, J. Freigang, S. Umhau, K. Zeth, J. Breed, Protein Sci. 7, 2413 (1998)], we predict that residues 142 to 494 contain 22 β strands. Thus, the structure of this portion of invasin may resemble those of the membrane-spanning portions of porins (16 to 18 β strands) [reviewed by B. K. Jap and P. J. Walian, Physiol. Rev. 76, 1073 (1996)] or FhuA (22 β strands) [K. P. Locher et al., Cell 95, 771 (1998); A. D. Ferguson, E. Hofmann, J. W. Coulton, K. Diederichs, W. Weite, Science 282, 2215 (1998)].
15. J. M. Leong, R. S. Foumier, R. R. Isberg, EMBO J. 9, 1979 (1990); M. J. Worley, I. Stojiljkovic, F. Heffron, Mol. Microbiol. 29, 1471 (1998). Using a neural network algorithm that predicts the membrane topology of integral outer membrane proteins [K. Diederichs, J. Freigang, S. Umhau, K. Zeth, J. Breed, Protein Sci. 7, 2413 (1998)], we predict that residues 142 to 494 contain 22 β strands. Thus, the structure of this portion of invasin may resemble those of the membrane-spanning portions of porins (16 to 18 β strands) [reviewed by B. K. Jap and P. J. Walian, Physiol. Rev. 76, 1073 (1996)] or FhuA (22 β strands) [K. P. Locher et al., Cell 95, 771 (1998); A. D. Ferguson, E. Hofmann, J. W. Coulton, K. Diederichs, W. Weite, Science 282, 2215 (1998)].
16. J. M. Leong, R. S. Foumier, R. R. Isberg, EMBO J. 9, 1979 (1990); M. J. Worley, I. Stojiljkovic, F. Heffron, Mol. Microbiol. 29, 1471 (1998). Using a neural network algorithm that predicts the membrane topology of integral outer membrane proteins [K. Diederichs, J. Freigang, S. Umhau, K. Zeth, J. Breed, Protein Sci. 7, 2413 (1998)], we predict that residues 142 to 494 contain 22 β strands. Thus, the structure of this portion of invasin may resemble those of the membrane-spanning portions of porins (16 to 18 β strands) [reviewed by B. K. Jap and P. J. Walian, Physiol. Rev. 76, 1073 (1996)] or FhuA (22 β strands) [K. P. Locher et al., Cell 95, 771 (1998); A. D. Ferguson, E. Hofmann, J. W. Coulton, K. Diederichs, W. Weite, Science 282, 2215 (1998)].
17. J. M. Leong, R. S. Foumier, R. R. Isberg, EMBO J. 9, 1979 (1990); M. J. Worley, I. Stojiljkovic, F. Heffron, Mol. Microbiol. 29, 1471 (1998). Using a neural network algorithm that predicts the membrane topology of integral outer membrane proteins [K. Diederichs, J. Freigang, S. Umhau, K. Zeth, J. Breed, Protein Sci. 7, 2413 (1998)], we predict that residues 142 to 494 contain 22 β strands. Thus, the structure of this portion of invasin may resemble those of the membrane-spanning portions of porins (16 to 18 β strands) [reviewed by B. K. Jap and P. J. Walian, Physiol. Rev. 76, 1073 (1996)] or FhuA (22 β strands) [K. P. Locher et al., Cell 95, 771 (1998); A. D. Ferguson, E. Hofmann, J. W. Coulton, K. Diederichs, W. Weite, Science 282, 2215 (1998)].
18. J. Yu and J. B. Kaber, Mol. Microbiol. 6, 411 (1992). A sequence identity of 30% has been established as the threshold for guaranteed three-dimensional similarity [C. Chothia and A. M. Lesk, EMBO J. 5, 823 (1986) ]. Length-dependent sequence identity thresholds are discussed in work by R. A. Abagyan and S. Batalov, J. Mol. Biol. 273, 355 (1997), and references therein. By these criteria, the cell-binding regions of invasin and intimin do not share significant sequence identity, and individual invasin domains do not share significant sequence similarity with Fn-III, IgSF, CRD, CTLD, or CTLD-related proteins.
19. J. Yu and J. B. Kaber, Mol. Microbiol. 6, 411 (1992). A sequence identity of 30% has been established as the threshold for guaranteed three-dimensional similarity [C. Chothia and A. M. Lesk, EMBO J. 5, 823 (1986) ]. Length-dependent sequence identity thresholds are discussed in work by R. A. Abagyan and S. Batalov, J. Mol. Biol. 273, 355 (1997), and references therein. By these criteria, the cell-binding regions of invasin and intimin do not share significant sequence identity, and individual invasin domains do not share significant sequence similarity with Fn-III, IgSF, CRD, CTLD, or CTLD-related proteins.
20. J. Yu and J. B. Kaber, Mol. Microbiol. 6, 411 (1992). A sequence identity of 30% has been established as the threshold for guaranteed three-dimensional similarity [C. Chothia and A. M. Lesk, EMBO J. 5, 823 (1986) ]. Length-dependent sequence identity thresholds are discussed in work by R. A. Abagyan and S. Batalov, J. Mol. Biol. 273, 355 (1997), and references therein. By these criteria, the cell-binding regions of invasin and intimin do not share significant sequence identity, and individual invasin domains do not share significant sequence similarity with Fn-III, IgSF, CRD, CTLD, or CTLD-related proteins.
21. 495, five residues shorter than the predicted site of cleavage (R. R. Isberg, unpublished data). A selenomethionine (SeMet)-substituted version of Inv497 was produced following the method of W. A. Hendrickson, J. R. Horton, D. M. LeMaster, EMBO J. 9, 1665 (1990), and purified under the same conditions as the native protein. Amino acid composition analysis showed ∼100% replacement of the eight methionines by SeMet (M. S. Brown and R. R. Isberg, data not shown)
22. 495, five residues shorter than the predicted site of cleavage (R. R. Isberg, unpublished data). A selenomethionine (SeMet)-substituted version of Inv497 was produced following the method of W. A. Hendrickson, J. R. Horton, D. M. LeMaster, EMBO J. 9, 1665 (1990), and purified under the same conditions as the native protein. Amino acid composition analysis showed ∼100% replacement of the eight methionines by SeMet (M. S. Brown and R. R. Isberg, data not shown)
23. 495, five residues shorter than the predicted site of cleavage (R. R. Isberg, unpublished data). A selenomethionine (SeMet)-substituted version of Inv497 was produced following the method of W. A. Hendrickson, J. R. Horton, D. M. LeMaster, EMBO J. 9, 1665 (1990), and purified under the same conditions as the native protein. Amino acid composition analysis showed ∼100% replacement of the eight methionines by SeMet (M. S. Brown and R. R. Isberg, data not shown)
24. 495, five residues shorter than the predicted site of cleavage (R. R. Isberg, unpublished data). A selenomethionine (SeMet)-substituted version of Inv497 was produced following the method of W. A. Hendrickson, J. R. Horton, D. M. LeMaster, EMBO J. 9, 1665 (1990), and purified under the same conditions as the native protein. Amino acid composition analysis showed ∼100% replacement of the eight methionines by SeMet (M. S. Brown and R. R. Isberg, data not shown)
25. G. Tran Van Nhieu and R. R. Isberg, J. Biol. Chem. 266, 24367 (1991); E. S. Krukonis, P. Dersch, J. A. Eble, R. R. Isberg, ibid. 273, 31837 (1998); J. A. Eble et al., Biochemistry 37, 10945 (1998); Y. Takada, J. Ylanne, D. Mandelman, W. Puzon, M. H. Ginsberg, J. Cell Biol. 119, 913 (1992).
26. G. Tran Van Nhieu and R. R. Isberg, J. Biol. Chem. 266, 24367 (1991); E. S. Krukonis, P. Dersch, J. A. Eble, R. R. Isberg, ibid. 273, 31837 (1998); J. A. Eble et al., Biochemistry 37, 10945 (1998); Y. Takada, J. Ylanne, D. Mandelman, W. Puzon, M. H. Ginsberg, J. Cell Biol. 119, 913 (1992).
27. G. Tran Van Nhieu and R. R. Isberg, J. Biol. Chem. 266, 24367 (1991); E. S. Krukonis, P. Dersch, J. A. Eble, R. R. Isberg, ibid. 273, 31837 (1998); J. A. Eble et al., Biochemistry 37, 10945 (1998); Y. Takada, J. Ylanne, D. Mandelman, W. Puzon, M. H. Ginsberg, J. Cell Biol. 119, 913 (1992).
28. G. Tran Van Nhieu and R. R. Isberg, J. Biol. Chem. 266, 24367 (1991); E. S. Krukonis, P. Dersch, J. A. Eble, R. R. Isberg, ibid. 273, 31837 (1998); J. A. Eble et al., Biochemistry 37, 10945 (1998); Y. Takada, J. Ylanne, D. Mandelman, W. Puzon, M. H. Ginsberg, J. Cell Biol. 119, 913 (1992).
29. Phasing and model building: A cryocooled xenon derivative was prepared with the apparatus described by S. M. Soltis, M. H. B. Stowell, M. C. Wiener, G. N. Phillips, D. C. Rees, J. Appl. Crystallogr. 30, 190 (1997). Data were processed and scaled with the HKL package [Z. Otwinowski and W. Minor, Methods Enzymol. 276, 307 (1997)]. Heavy-atom refinement and phasing were performed with the program SHARP [E. De La Fortelle and G. Bricogne, Methods Enzymol. 276, 472 (1997)]. Difference Patterson maps for the xenon and di-μ-iodobis(ethylenediamine) diplatinum nitrate (PIP) derivatives were interpreted with XTALVIEW [D. E. McRee, Practical Protein Crystallography (Academic Press, San Diego, CA, 1993)], and one xenon, three platinum, and two iodine sites were refined with SHARP. An initial MIRAS electron density map was calculated to 3.6 Å and solvent flattened with Solomon [J. P. Abrahams and A. G. W. Leslie, Acta Crystallogr. D52, 30 (1996)] as implemented in SHARP. A skeleton of the map [G. J. Kleywegt and T. A. Jones, Acta Crystallogr. D52, 826 (1997)] served as a starting point for model building with the program O (22). The initial electron density map revealed the Ig-like domain structures of the first four domains, but a definitive assignment of side chains was not possible, and the connectivity in D5 was ambiguous. Using MIRAS phases, we found eight selenium sites in a difference Fourier map calculated for the SeMet derivative, which allowed identification of methionines that were used as markers for the assignment of the rest of the sequence. After including the SeMet sites in heavy-atom positional refinement using SHARP, we calculated an improved solvent-flattened MIRAS electron density map to 3.2 Å resolution with a mean figure of merit of 0.509.
30. Phasing and model building: A cryocooled xenon derivative was prepared with the apparatus described by S. M. Soltis, M. H. B. Stowell, M. C. Wiener, G. N. Phillips, D. C. Rees, J. Appl. Crystallogr. 30, 190 (1997). Data were processed and scaled with the HKL package [Z. Otwinowski and W. Minor, Methods Enzymol. 276, 307 (1997)]. Heavy-atom refinement and phasing were performed with the program SHARP [E. De La Fortelle and G. Bricogne, Methods Enzymol. 276, 472 (1997)]. Difference Patterson maps for the xenon and di-μ-iodobis(ethylenediamine) diplatinum nitrate (PIP) derivatives were interpreted with XTALVIEW [D. E. McRee, Practical Protein Crystallography (Academic Press, San Diego, CA, 1993)], and one xenon, three platinum, and two iodine sites were refined with SHARP. An initial MIRAS electron density map was calculated to 3.6 Å and solvent flattened with Solomon [J. P. Abrahams and A. G. W. Leslie, Acta Crystallogr. D52, 30 (1996)] as implemented in SHARP. A skeleton of the map [G. J. Kleywegt and T. A. Jones, Acta Crystallogr. D52, 826 (1997)] served as a starting point for model building with the program O (22). The initial electron density map revealed the Ig-like domain structures of the first four domains, but a definitive assignment of side chains was not possible, and the connectivity in D5 was ambiguous. Using MIRAS phases, we found eight selenium sites in a difference Fourier map calculated for the SeMet derivative, which allowed identification of methionines that were used as markers for the assignment of the rest of the sequence. After including the SeMet sites in heavy-atom positional refinement using SHARP, we calculated an improved solvent-flattened MIRAS electron density map to 3.2 Å resolution with a mean figure of merit of 0.509.
31. Phasing and model building: A cryocooled xenon derivative was prepared with the apparatus described by S. M. Soltis, M. H. B. Stowell, M. C. Wiener, G. N. Phillips, D. C. Rees, J. Appl. Crystallogr. 30, 190 (1997). Data were processed and scaled with the HKL package [Z. Otwinowski and W. Minor, Methods Enzymol. 276, 307 (1997)]. Heavy-atom refinement and phasing were performed with the program SHARP [E. De La Fortelle and G. Bricogne, Methods Enzymol. 276, 472 (1997)]. Difference Patterson maps for the xenon and di-μ-iodobis(ethylenediamine) diplatinum nitrate (PIP) derivatives were interpreted with XTALVIEW [D. E. McRee, Practical Protein Crystallography (Academic Press, San Diego, CA, 1993)], and one xenon, three platinum, and two iodine sites were refined with SHARP. An initial MIRAS electron density map was calculated to 3.6 Å and solvent flattened with Solomon [J. P. Abrahams and A. G. W. Leslie, Acta Crystallogr. D52, 30 (1996)] as implemented in SHARP. A skeleton of the map [G. J. Kleywegt and T. A. Jones, Acta Crystallogr. D52, 826 (1997)] served as a starting point for model building with the program O (22). The initial electron density map revealed the Ig-like domain structures of the first four domains, but a definitive assignment of side chains was not possible, and the connectivity in D5 was ambiguous. Using MIRAS phases, we found eight selenium sites in a difference Fourier map calculated for the SeMet derivative, which allowed identification of methionines that were used as markers for the assignment of the rest of the sequence. After including the SeMet sites in heavy-atom positional refinement using SHARP, we calculated an improved solvent-flattened MIRAS electron density map to 3.2 Å resolution with a mean figure of merit of 0.509.
32. Phasing and model building: A cryocooled xenon derivative was prepared with the apparatus described by S. M. Soltis, M. H. B. Stowell, M. C. Wiener, G. N. Phillips, D. C. Rees, J. Appl. Crystallogr. 30, 190 (1997). Data were processed and scaled with the HKL package [Z. Otwinowski and W. Minor, Methods Enzymol. 276, 307 (1997)]. Heavy-atom refinement and phasing were performed with the program SHARP [E. De La Fortelle and G. Bricogne, Methods Enzymol. 276, 472 (1997)]. Difference Patterson maps for the xenon and di-μ-iodobis(ethylenediamine) diplatinum nitrate (PIP) derivatives were interpreted with XTALVIEW [D. E. McRee, Practical Protein Crystallography (Academic Press, San Diego, CA, 1993)], and one xenon, three platinum, and two iodine sites were refined with SHARP. An initial MIRAS electron density map was calculated to 3.6 Å and solvent flattened with Solomon [J. P. Abrahams and A. G. W. Leslie, Acta Crystallogr. D52, 30 (1996)] as implemented in SHARP. A skeleton of the map [G. J. Kleywegt and T. A. Jones, Acta Crystallogr. D52, 826 (1997)] served as a starting point for model building with the program O (22). The initial electron density map revealed the Ig-like domain structures of the first four domains, but a definitive assignment of side chains was not possible, and the connectivity in D5 was ambiguous. Using MIRAS phases, we found eight selenium sites in a difference Fourier map calculated for the SeMet derivative, which allowed identification of methionines that were used as markers for the assignment of the rest of the sequence. After including the SeMet sites in heavy-atom positional refinement using SHARP, we calculated an improved solvent-flattened MIRAS electron density map to 3.2 Å resolution with a mean figure of merit of 0.509.
33. Phasing and model building: A cryocooled xenon derivative was prepared with the apparatus described by S. M. Soltis, M. H. B. Stowell, M. C. Wiener, G. N. Phillips, D. C. Rees, J. Appl. Crystallogr. 30, 190 (1997). Data were processed and scaled with the HKL package [Z. Otwinowski and W. Minor, Methods Enzymol. 276, 307 (1997)]. Heavy-atom refinement and phasing were performed with the program SHARP [E. De La Fortelle and G. Bricogne, Methods Enzymol. 276, 472 (1997)]. Difference Patterson maps for the xenon and di-μ-iodobis(ethylenediamine) diplatinum nitrate (PIP) derivatives were interpreted with XTALVIEW [D. E. McRee, Practical Protein Crystallography (Academic Press, San Diego, CA, 1993)], and one xenon, three platinum, and two iodine sites were refined with SHARP. An initial MIRAS electron density map was calculated to 3.6 Å and solvent flattened with Solomon [J. P. Abrahams and A. G. W. Leslie, Acta Crystallogr. D52, 30 (1996)] as implemented in SHARP. A skeleton of the map [G. J. Kleywegt and T. A. Jones, Acta Crystallogr. D52, 826 (1997)] served as a starting point for model building with the program O (22). The initial electron density map revealed the Ig-like domain structures of the first four domains, but a definitive assignment of side chains was not possible, and the connectivity in D5 was ambiguous. Using MIRAS phases, we found eight selenium sites in a difference Fourier map calculated for the SeMet derivative, which allowed identification of methionines that were used as markers for the assignment of the rest of the sequence. After including the SeMet sites in heavy-atom positional refinement using SHARP, we calculated an improved solvent-flattened MIRAS electron density map to 3.2 Å resolution with a mean figure of merit of 0.509.
34. Phasing and model building: A cryocooled xenon derivative was prepared with the apparatus described by S. M. Soltis, M. H. B. Stowell, M. C. Wiener, G. N. Phillips, D. C. Rees, J. Appl. Crystallogr. 30, 190 (1997). Data were processed and scaled with the HKL package [Z. Otwinowski and W. Minor, Methods Enzymol. 276, 307 (1997)]. Heavy-atom refinement and phasing were performed with the program SHARP [E. De La Fortelle and G. Bricogne, Methods Enzymol. 276, 472 (1997)]. Difference Patterson maps for the xenon and di-μ-iodobis(ethylenediamine) diplatinum nitrate (PIP) derivatives were interpreted with XTALVIEW [D. E. McRee, Practical Protein Crystallography (Academic Press, San Diego, CA, 1993)], and one xenon, three platinum, and two iodine sites were refined with SHARP. An initial MIRAS electron density map was calculated to 3.6 Å and solvent flattened with Solomon [J. P. Abrahams and A. G. W. Leslie, Acta Crystallogr. D52, 30 (1996)] as implemented in SHARP. A skeleton of the map [G. J. Kleywegt and T. A. Jones, Acta Crystallogr. D52, 826 (1997)] served as a starting point for model building with the program O (22). The initial electron density map revealed the Ig-like domain structures of the first four domains, but a definitive assignment of side chains was not possible, and the connectivity in D5 was ambiguous. Using MIRAS phases, we found eight selenium sites in a difference Fourier map calculated for the SeMet derivative, which allowed identification of methionines that were used as markers for the assignment of the rest of the sequence. After including the SeMet sites in heavy-atom positional refinement using SHARP, we calculated an improved solvent-flattened MIRAS electron density map to 3.2 Å resolution with a mean figure of merit of 0.509.
35. 2. Several regions include residues with real space correlation values (22) below 1σ from the mean (residues 531 to 534, 582 to 586, 647 to 650, 676 to 679, 779 to 780, 892 to 899, 955 to 957, and 969 to 977). Ramachandran plot statistics (Table 1) are as defined by PROCHECK [R. A. Laskowski, M. W. McArthur, D. S. Moss, J. M. Thornton, J. Appl. Crystallogr. 26, 283 (1993)]. Figures were made with MOLSCRIPT [P. J. Kraulis, J. Appl. Crystallogr. 24, 946 (1991)] and RASTER-3D [E. A. Merritt and M. E. P. Murphy, Acta Cystallogr. D50, 869 (1994)]. Molecular surfaces were generated with GRASP [A. Nicholls, R. Bharadwaj, B. Honig, Biophys. J. 64, A166 (1993)].
36. 2. Several regions include residues with real space correlation values (22) below 1σ from the mean (residues 531 to 534, 582 to 586, 647 to 650, 676 to 679, 779 to 780, 892 to 899, 955 to 957, and 969 to 977). Ramachandran plot statistics (Table 1) are as defined by PROCHECK [R. A. Laskowski, M. W. McArthur, D. S. Moss, J. M. Thornton, J. Appl. Crystallogr. 26, 283 (1993)]. Figures were made with MOLSCRIPT [P. J. Kraulis, J. Appl. Crystallogr. 24, 946 (1991)] and RASTER-3D [E. A. Merritt and M. E. P. Murphy, Acta Cystallogr. D50, 869 (1994)]. Molecular surfaces were generated with GRASP [A. Nicholls, R. Bharadwaj, B. Honig, Biophys. J. 64, A166 (1993)].
37. 2. Several regions include residues with real space correlation values (22) below 1σ from the mean (residues 531 to 534, 582 to 586, 647 to 650, 676 to 679, 779 to 780, 892 to 899, 955 to 957, and 969 to 977). Ramachandran plot statistics (Table 1) are as defined by PROCHECK [R. A. Laskowski, M. W. McArthur, D. S. Moss, J. M. Thornton, J. Appl. Crystallogr. 26, 283 (1993)]. Figures were made with MOLSCRIPT [P. J. Kraulis, J. Appl. Crystallogr. 24, 946 (1991)] and RASTER-3D [E. A. Merritt and M. E. P. Murphy, Acta Cystallogr. D50, 869 (1994)]. Molecular surfaces were generated with GRASP [A. Nicholls, R. Bharadwaj, B. Honig, Biophys. J. 64, A166 (1993)].
38. 2. Several regions include residues with real space correlation values (22) below 1σ from the mean (residues 531 to 534, 582 to 586, 647 to 650, 676 to 679, 779 to 780, 892 to 899, 955 to 957, and 969 to 977). Ramachandran plot statistics (Table 1) are as defined by PROCHECK [R. A. Laskowski, M. W. McArthur, D. S. Moss, J. M. Thornton, J. Appl. Crystallogr. 26, 283 (1993)]. Figures were made with MOLSCRIPT [P. J. Kraulis, J. Appl. Crystallogr. 24, 946 (1991)] and RASTER-3D [E. A. Merritt and M. E. P. Murphy, Acta Cystallogr. D50, 869 (1994)]. Molecular surfaces were generated with GRASP [A. Nicholls, R. Bharadwaj, B. Honig, Biophys. J. 64, A166 (1993)].
39. 2. Several regions include residues with real space correlation values (22) below 1σ from the mean (residues 531 to 534, 582 to 586, 647 to 650, 676 to 679, 779 to 780, 892 to 899, 955 to 957, and 969 to 977). Ramachandran plot statistics (Table 1) are as defined by PROCHECK [R. A. Laskowski, M. W. McArthur, D. S. Moss, J. M. Thornton, J. Appl. Crystallogr. 26, 283 (1993)]. Figures were made with MOLSCRIPT [P. J. Kraulis, J. Appl. Crystallogr. 24, 946 (1991)] and RASTER-3D [E. A. Merritt and M. E. P. Murphy, Acta Cystallogr. D50, 869 (1994)]. Molecular surfaces were generated with GRASP [A. Nicholls, R. Bharadwaj, B. Honig, Biophys. J. 64, A166 (1993)].
40. 2. Several regions include residues with real space correlation values (22) below 1σ from the mean (residues 531 to 534, 582 to 586, 647 to 650, 676 to 679, 779 to 780, 892 to 899, 955 to 957, and 969 to 977). Ramachandran plot statistics (Table 1) are as defined by PROCHECK [R. A. Laskowski, M. W. McArthur, D. S. Moss, J. M. Thornton, J. Appl. Crystallogr. 26, 283 (1993)]. Figures were made with MOLSCRIPT [P. J. Kraulis, J. Appl. Crystallogr. 24, 946 (1991)] and RASTER-3D [E. A. Merritt and M. E. P. Murphy, Acta Cystallogr. D50, 869 (1994)]. Molecular surfaces were generated with GRASP [A. Nicholls, R. Bharadwaj, B. Honig, Biophys. J. 64, A166 (1993)].
41. 2. Several regions include residues with real space correlation values (22) below 1σ from the mean (residues 531 to 534, 582 to 586, 647 to 650, 676 to 679, 779 to 780, 892 to 899, 955 to 957, and 969 to 977). Ramachandran plot statistics (Table 1) are as defined by PROCHECK [R. A. Laskowski, M. W. McArthur, D. S. Moss, J. M. Thornton, J. Appl. Crystallogr. 26, 283 (1993)]. Figures were made with MOLSCRIPT [P. J. Kraulis, J. Appl. Crystallogr. 24, 946 (1991)] and RASTER-3D [E. A. Merritt and M. E. P. Murphy, Acta Cystallogr. D50, 869 (1994)]. Molecular surfaces were generated with GRASP [A. Nicholls, R. Bharadwaj, B. Honig, Biophys. J. 64, A166 (1993)].
42. 2. Several regions include residues with real space correlation values (22) below 1σ from the mean (residues 531 to 534, 582 to 586, 647 to 650, 676 to 679, 779 to 780, 892 to 899, 955 to 957, and 969 to 977). Ramachandran plot statistics (Table 1) are as defined by PROCHECK [R. A. Laskowski, M. W. McArthur, D. S. Moss, J. M. Thornton, J. Appl. Crystallogr. 26, 283 (1993)]. Figures were made with MOLSCRIPT [P. J. Kraulis, J. Appl. Crystallogr. 24, 946 (1991)] and RASTER-3D [E. A. Merritt and M. E. P. Murphy, Acta Cystallogr. D50, 869 (1994)]. Molecular surfaces were generated with GRASP [A. Nicholls, R. Bharadwaj, B. Honig, Biophys. J. 64, A166 (1993)].
43. Equilibrium analytical ultracentrifugation analyses establish that Inv497 is monomeric at micromolar concentrations in solution [P. Dersch and R. R. Isberg, EMBO J. 18, 1199 (1999)]. Sedimentation velocity analytical uttracentrifugation experiments suggest that Inv497 is elongated in solution [X.-D. Su et al., Science 281, 991 (1998)]; thus, the extended conformation does not result from crystal packing forces.
44. Equilibrium analytical ultracentrifugation analyses establish that Inv497 is monomeric at micromolar concentrations in solution [P. Dersch and R. R. Isberg, EMBO J. 18, 1199 (1999)]. Sedimentation velocity analytical uttracentrifugation experiments suggest that Inv497 is elongated in solution [X.-D. Su et al., Science 281, 991 (1998)]; thus, the extended conformation does not result from crystal packing forces.
45. Protein structures: Fn-III 7-10 [Protein Data Bank (PDB) code 1FNF] [D. J. Leahy, I. Aukhil, H. P. Erickson, Cell 84, 155 (1996)]; Fn-III 12-14 (PDB code 1FNH) [A. Sharma, J. A. Askari, M. J. Humphries, E. Y. Jones, D. I. Stuart, EMBO J. 18, 1468 (1999)]; intimin (coordinates obtained from S. Matthews) [G. Kelly et al., Nature Struct Biol. 6, 313 (1999)]; mannose-binding protein (PDB code 1RTM) [W. I. Weis and K. Drickamer, Structure 2, 1227 (1994)]; E-selectin (PDB code 1ESL) [B. J. Graves et al., Nature 367, 532 (1994)]; (coordinates obtained from P. D. Sun) [J. C. Boyington et al., Immunity 10, 75 (1999)]; VCAM-1 (PDB code 1VSC) [E. Y. Jones et al., Nature 373, 539 (1995); J. Wang et al., Proc. Natl. Acad. Sci. U.S.A. 92, 5714 (1995)]; and ICAM-1 (PDB code 1IC1) [(23); J. Bella, P. R. Kolatkar, C. W. Marlor, J. M. Greve, M. G. Rossmann, Proc Natl. Acad. Sci. U.S.A. 95, 4140 (1998)].
46. Protein structures: Fn-III 7-10 [Protein Data Bank (PDB) code 1FNF] [D. J. Leahy, I. Aukhil, H. P. Erickson, Cell 84, 155 (1996)]; Fn-III 12-14 (PDB code 1FNH) [A. Sharma, J. A. Askari, M. J. Humphries, E. Y. Jones, D. I. Stuart, EMBO J. 18, 1468 (1999)]; intimin (coordinates obtained from S. Matthews) [G. Kelly et al., Nature Struct Biol. 6, 313 (1999)]; mannose-binding protein (PDB code 1RTM) [W. I. Weis and K. Drickamer, Structure 2, 1227 (1994)]; E-selectin (PDB code 1ESL) [B. J. Graves et al., Nature 367, 532 (1994)]; (coordinates obtained from P. D. Sun) [J. C. Boyington et al., Immunity 10, 75 (1999)]; VCAM-1 (PDB code 1VSC) [E. Y. Jones et al., Nature 373, 539 (1995); J. Wang et al., Proc. Natl. Acad. Sci. U.S.A. 92, 5714 (1995)]; and ICAM-1 (PDB code 1IC1) [(23); J. Bella, P. R. Kolatkar, C. W. Marlor, J. M. Greve, M. G. Rossmann, Proc Natl. Acad. Sci. U.S.A. 95, 4140 (1998)].
47. Protein structures: Fn-III 7-10 [Protein Data Bank (PDB) code 1FNF] [D. J. Leahy, I. Aukhil, H. P. Erickson, Cell 84, 155 (1996)]; Fn-III 12-14 (PDB code 1FNH) [A. Sharma, J. A. Askari, M. J. Humphries, E. Y. Jones, D. I. Stuart, EMBO J. 18, 1468 (1999)]; intimin (coordinates obtained from S. Matthews) [G. Kelly et al., Nature Struct Biol. 6, 313 (1999)]; mannose-binding protein (PDB code 1RTM) [W. I. Weis and K. Drickamer, Structure 2, 1227 (1994)]; E-selectin (PDB code 1ESL) [B. J. Graves et al., Nature 367, 532 (1994)]; (coordinates obtained from P. D. Sun) [J. C. Boyington et al., Immunity 10, 75 (1999)]; VCAM-1 (PDB code 1VSC) [E. Y. Jones et al., Nature 373, 539 (1995); J. Wang et al., Proc. Natl. Acad. Sci. U.S.A. 92, 5714 (1995)]; and ICAM-1 (PDB code 1IC1) [(23); J. Bella, P. R. Kolatkar, C. W. Marlor, J. M. Greve, M. G. Rossmann, Proc Natl. Acad. Sci. U.S.A. 95, 4140 (1998)].
48. Protein structures: Fn-III 7-10 [Protein Data Bank (PDB) code 1FNF] [D. J. Leahy, I. Aukhil, H. P. Erickson, Cell 84, 155 (1996)]; Fn-III 12-14 (PDB code 1FNH) [A. Sharma, J. A. Askari, M. J. Humphries, E. Y. Jones, D. I. Stuart, EMBO J. 18, 1468 (1999)]; intimin (coordinates obtained from S. Matthews) [G. Kelly et al., Nature Struct Biol. 6, 313 (1999)]; mannose-binding protein (PDB code 1RTM) [W. I. Weis and K. Drickamer, Structure 2, 1227 (1994)]; E-selectin (PDB code 1ESL) [B. J. Graves et al., Nature 367, 532 (1994)]; (coordinates obtained from P. D. Sun) [J. C. Boyington et al., Immunity 10, 75 (1999)]; VCAM-1 (PDB code 1VSC) [E. Y. Jones et al., Nature 373, 539 (1995); J. Wang et al., Proc. Natl. Acad. Sci. U.S.A. 92, 5714 (1995)]; and ICAM-1 (PDB code 1IC1) [(23); J. Bella, P. R. Kolatkar, C. W. Marlor, J. M. Greve, M. G. Rossmann, Proc Natl. Acad. Sci. U.S.A. 95, 4140 (1998)].
49. Protein structures: Fn-III 7-10 [Protein Data Bank (PDB) code 1FNF] [D. J. Leahy, I. Aukhil, H. P. Erickson, Cell 84, 155 (1996)]; Fn-III 12-14 (PDB code 1FNH) [A. Sharma, J. A. Askari, M. J. Humphries, E. Y. Jones, D. I. Stuart, EMBO J. 18, 1468 (1999)]; intimin (coordinates obtained from S. Matthews) [G. Kelly et al., Nature Struct Biol. 6, 313 (1999)]; mannose-binding protein (PDB code 1RTM) [W. I. Weis and K. Drickamer, Structure 2, 1227 (1994)]; E-selectin (PDB code 1ESL) [B. J. Graves et al., Nature 367, 532 (1994)]; (coordinates obtained from P. D. Sun) [J. C. Boyington et al., Immunity 10, 75 (1999)]; VCAM-1 (PDB code 1VSC) [E. Y. Jones et al., Nature 373, 539 (1995); J. Wang et al., Proc. Natl. Acad. Sci. U.S.A. 92, 5714 (1995)]; and ICAM-1 (PDB code 1IC1) [(23); J. Bella, P. R. Kolatkar, C. W. Marlor, J. M. Greve, M. G. Rossmann, Proc Natl. Acad. Sci. U.S.A. 95, 4140 (1998)].
50. Protein structures: Fn-III 7-10 [Protein Data Bank (PDB) code 1FNF] [D. J. Leahy, I. Aukhil, H. P. Erickson, Cell 84, 155 (1996)]; Fn-III 12-14 (PDB code 1FNH) [A. Sharma, J. A. Askari, M. J. Humphries, E. Y. Jones, D. I. Stuart, EMBO J. 18, 1468 (1999)]; intimin (coordinates obtained from S. Matthews) [G. Kelly et al., Nature Struct Biol. 6, 313 (1999)]; mannose-binding protein (PDB code 1RTM) [W. I. Weis and K. Drickamer, Structure 2, 1227 (1994)]; E-selectin (PDB code 1ESL) [B. J. Graves et al., Nature 367, 532 (1994)]; (coordinates obtained from P. D. Sun) [J. C. Boyington et al., Immunity 10, 75 (1999)]; VCAM-1 (PDB code 1VSC) [E. Y. Jones et al., Nature 373, 539 (1995); J. Wang et al., Proc. Natl. Acad. Sci. U.S.A. 92, 5714 (1995)]; and ICAM-1 (PDB code 1IC1) [(23); J. Bella, P. R. Kolatkar, C. W. Marlor, J. M. Greve, M. G. Rossmann, Proc Natl. Acad. Sci. U.S.A. 95, 4140 (1998)].
51. Protein structures: Fn-III 7-10 [Protein Data Bank (PDB) code 1FNF] [D. J. Leahy, I. Aukhil, H. P. Erickson, Cell 84, 155 (1996)]; Fn-III 12-14 (PDB code 1FNH) [A. Sharma, J. A. Askari, M. J. Humphries, E. Y. Jones, D. I. Stuart, EMBO J. 18, 1468 (1999)]; intimin (coordinates obtained from S. Matthews) [G. Kelly et al., Nature Struct Biol. 6, 313 (1999)]; mannose-binding protein (PDB code 1RTM) [W. I. Weis and K. Drickamer, Structure 2, 1227 (1994)]; E-selectin (PDB code 1ESL) [B. J. Graves et al., Nature 367, 532 (1994)]; (coordinates obtained from P. D. Sun) [J. C. Boyington et al., Immunity 10, 75 (1999)]; VCAM-1 (PDB code 1VSC) [E. Y. Jones et al., Nature 373, 539 (1995); J. Wang et al., Proc. Natl. Acad. Sci. U.S.A. 92, 5714 (1995)]; and ICAM-1 (PDB code 1IC1) [(23); J. Bella, P. R. Kolatkar, C. W. Marlor, J. M. Greve, M. G. Rossmann, Proc Natl. Acad. Sci. U.S.A. 95, 4140 (1998)].
52. Protein structures: Fn-III 7-10 [Protein Data Bank (PDB) code 1FNF] [D. J. Leahy, I. Aukhil, H. P. Erickson, Cell 84, 155 (1996)]; Fn-III 12-14 (PDB code 1FNH) [A. Sharma, J. A. Askari, M. J. Humphries, E. Y. Jones, D. I. Stuart, EMBO J. 18, 1468 (1999)]; intimin (coordinates obtained from S. Matthews) [G. Kelly et al., Nature Struct Biol. 6, 313 (1999)]; mannose-binding protein (PDB code 1RTM) [W. I. Weis and K. Drickamer, Structure 2, 1227 (1994)]; E-selectin (PDB code 1ESL) [B. J. Graves et al., Nature 367, 532 (1994)]; (coordinates obtained from P. D. Sun) [J. C. Boyington et al., Immunity 10, 75 (1999)]; VCAM-1 (PDB code 1VSC) [E. Y. Jones et al., Nature 373, 539 (1995); J. Wang et al., Proc. Natl. Acad. Sci. U.S.A. 92, 5714 (1995)]; and ICAM-1 (PDB code 1IC1) [(23); J. Bella, P. R. Kolatkar, C. W. Marlor, J. M. Greve, M. G. Rossmann, Proc Natl. Acad. Sci. U.S.A. 95, 4140 (1998)].
53. Protein structures: Fn-III 7-10 [Protein Data Bank (PDB) code 1FNF] [D. J. Leahy, I. Aukhil, H. P. Erickson, Cell 84, 155 (1996)]; Fn-III 12-14 (PDB code 1FNH) [A. Sharma, J. A. Askari, M. J. Humphries, E. Y. Jones, D. I. Stuart, EMBO J. 18, 1468 (1999)]; intimin (coordinates obtained from S. Matthews) [G. Kelly et al., Nature Struct Biol. 6, 313 (1999)]; mannose-binding protein (PDB code 1RTM) [W. I. Weis and K. Drickamer, Structure 2, 1227 (1994)]; E-selectin (PDB code 1ESL) [B. J. Graves et al., Nature 367, 532 (1994)]; (coordinates obtained from P. D. Sun) [J. C. Boyington et al., Immunity 10, 75 (1999)]; VCAM-1 (PDB code 1VSC) [E. Y. Jones et al., Nature 373, 539 (1995); J. Wang et al., Proc. Natl. Acad. Sci. U.S.A. 92, 5714 (1995)]; and ICAM-1 (PDB code 1IC1) [(23); J. Bella, P. R. Kolatkar, C. W. Marlor, J. M. Greve, M. G. Rossmann, Proc Natl. Acad. Sci. U.S.A. 95, 4140 (1998)].
54. IgSF domains were previously classified into V, C1, C2, 11, and 12 sets on the basis of similarities in sequence and structure [Y. Harpaz and C. Chothia, J. Mol. Biol. 238, 528 (1994); (23)]. The V and C1 sets are similar to antibody variable and constant domains, respectively. The V set consists of two β sheets: one containing β strands ABED and the other containing strands A'GFCC'C'. The C1 set contains an ABED and a GFC sheet. The two sheets of the C2 set are ABE and GFCC'. The I set domains are intermediate between the V and C1 sets. The I1 set contains ABED and A'GFC sheets, and the I2 set contains ABE and A'GFCC' sheets. D1 through D4 of Inv497 adopt folding topologies that resemble IgSF domains but lack the core residues and disulfide bonds conserved in IgSF members.
55. 935).
56. 935).
57. Although direct binding of invasin to a carbohydrate has not been demonstrated, high concentrations of N-acetylneuraminic acid [median inhibitory concentration of 20 mM) inhibit mammalian cell adhesion to immobilized invasin. A variety of other acetylated sugars showed no such inhibition (R. R. Isberg, unpublished results).
58. Interrupted α helices have been observed in other protein structures, including subtilisin [reviewed by J. S. Richardson, Adv. Protein Chem. 34, 167 (1981)] and fibritin [Y. Tao, S. V. Strelkov, V. V. Mesyanzhinov, M. G. Rossmann, Structure 5, 789 (1997)].
59. Interrupted α helices have been observed in other protein structures, including subtilisin [reviewed by J. S. Richardson, Adv. Protein Chem. 34, 167 (1981)] and fibritin [Y. Tao, S. V. Strelkov, V. V. Mesyanzhinov, M. G. Rossmann, Structure 5, 789 (1997)].
60. 2.
61. J. M. Leong, P. E. Morrissey, R. R. Isberg, J. Biol. Chem. 268, 20524 (1993); L. H. Saltman, Y. Lu, E. M. Zaharias, R. R. Isberg, ibid. 271, 23438 (1996).
62. J. M. Leong, P. E. Morrissey, R. R. Isberg, J. Biol. Chem. 268, 20524 (1993); L. H. Saltman, Y. Lu, E. M. Zaharias, R. R. Isberg, ibid. 271, 23438 (1996).
63. R. D. Bowditch et al., ibid. 269, 10856 (1994); S.-I. Aota, M. Nomizu, K. M. Yamada, ibid., p. 24756; T. P. Ugarova et al., Biochemistry 34, 4457 (1995).
64. R. D. Bowditch et al., ibid. 269, 10856 (1994); S.-I. Aota, M. Nomizu, K. M. Yamada, ibid., p. 24756; T. P. Ugarova et al., Biochemistry 34, 4457 (1995).
65. R. D. Bowditch et al., ibid. 269, 10856 (1994); S.-I. Aota, M. Nomizu, K. M. Yamada, ibid., p. 24756; T. P. Ugarova et al., Biochemistry 34, 4457 (1995).
66. T. A. Jones and M. Kjeldgaard, Methods Enzymol. 277, 173 (1997).
67. J. M. Casasnovas, T. Stehle, J. Liu, J. Wang, T. A. Springer, Proc. Natl. Acad. Sci. U.S.A. 95, 4134 (1998).
68. Single-letter abbreviations for the amino acid residues are as follows: A, Ala; D, Asp; G, Gly; L, Leu; N, Asn; P, Pro; R, Arg; T, Thr; and W, Trp.
69. R. R. Isberg, Y. Yang, D. L. Voorhis, J. Biol. Chem. 268, 15840 (1993).
70. We thank S. M. Soltis and the staff at the Stanford Synchrotron Radiation Laboratory (SSRL) for help with xenon derivatization and data collection; M. J. Bennett, A. J. Chirino, L M. Sánchez, D. E. Vaughn, and A. P. Yeh for discussions and help with crystallographic software; S. Matthews for intimin coordinates; P. D. Sun for CD94 coordinates; W. I. Weis for helpful discussions about C-type lectin structures; and W. I Weis, J. M. Leong, and members of the Bjorkman lab for critical reading of the manuscript Inv497 coordinates have been deposited in the PDB (PDB code 1CWV).