High-resolution solution NMR structure of the Z domain of staphylococcal protein A.

Staphylococcal protein A (SpA) is a cell-wall-bound pathogenicity factor from the bacterium Staphylococcus aureus. Because of their small size and immunoglobulin (IgG)-binding activities, domains of protein A are targets for protein engineering efforts and for the development of computational approaches for de novo protein folding. The NMR solution structure of an engineered IgG-binding domain of SpA, the Z domain (an analog of the B domain of SpA), has been determined by simulated annealing with restrained molecular dynamics on the basis of 671 conformational constraints. The Z domain contains three well-defined alpha-helices corresponding to polypeptide segments Lys7 to Leu17 (helix 1), Glu24 to Asp36 (helix 2), and Ser41 to Ala54 (helix 3). A family of ten conformers representing the solution structure of the Z domain was computed by simulated annealing of restrained molecular dynamics using the program CONGEN. The average of the root-mean-square deviations (r.m. s.d.) of the individual NMR conformers, relative to the mean coordinates, for the backbone atoms N, Calpha and C' of residues Phe5 through Ala56 is 0.69 A; the corresponding backbone r.m.s.d. for the three-helical core is 0.44 A. Helices 1, 2 and 3 are antiparallel in orientation (Omega12=-170(+/-4) degrees , Omega13=+16(+/-3) degrees , Omega23=+173(+/-7) degrees ). A comparison of backbone amide hydrogen/deuterium exchange rates in free and IgG-bound Z domains demonstrates that the amide protons of helices 1, 2 and 3 are protected from rapid exchange in both states, indicating that all three helices are also intact in the IgG-bound state. These solution NMR results differ from the previously determined X-ray structure of the similar SpA B domain in complex with the Fc fragment of a human IgG antibody, where helix 3 is not observed in the electron density map and from the solution NMR structure of the B domain, where helix 3 is observed but helix 1 is tilted by approximately 30 degrees with respect to helices 2 and 3. Hydrogen-bonded N-cap and C-cap formation is observed for all three helices of the Z domain; these capping interactions appear to be highly conserved in the five homologous domains of SpA.

[1]  G. Montelione,et al.  Classification of amino acid spin systems using PFG HCC(CO)NH-TOCSY with constant-time aliphatic 13C frequency labeling , 1995, Journal of biomolecular NMR.

[2]  Roberto Tejero,et al.  Simulated annealing with restrained molecular dynamics using a flexible restraint potential: Theory and evaluation with simulated NMR constraints , 1996, Protein science : a publication of the Protein Society.

[3]  R. Huber,et al.  Crystallographic structural studies of a human Fc fragment. II. A complete model based on a Fourier map at 3.5 A resolution. , 1976, Hoppe-Seyler's Zeitschrift fur physiologische Chemie.

[4]  M. Uhlén,et al.  Staphylococcal protein A consists of five IgG-binding domains. , 1986, European journal of biochemistry.

[5]  I. Shimada,et al.  Three-dimensional solution structure of the B domain of staphylococcal protein A: comparisons of the solution and crystal structures. , 1992, Biochemistry.

[6]  A. Gronenborn,et al.  Identification of N-terminal helix capping boxes by means of 13C chemical shifts , 1994, Journal of biomolecular NMR.

[7]  C. Jenkin,et al.  Isolation of pure IgG1, IgG2a and IgG2b immunoglobulins from mouse serum using protein A-sepharose. , 1978, Immunochemistry.

[8]  J. Skolnick,et al.  Monte carlo simulations of protein folding. II. Application to protein A, ROP, and crambin , 1994, Proteins.

[9]  I. Shimada,et al.  Sequential 1H NMR assignments and secondary structure of the B domain of staphylococcal protein A: structural changes between the free B domain in solution and the Fc-bound B domain in crystal. , 1990, Biochemistry.

[10]  A. Forsgren,et al.  “Protein A” from S. Aureus I. Pseudo-Immune Reaction with Human γ-Globulin , 1966 .

[11]  A Wlodawer,et al.  Structural comparisons among the short-chain helical cytokines. , 1994, Structure.

[12]  A. Gronenborn,et al.  Complete resonance assignment for the polypeptide backbone of interleukin 1 beta using three-dimensional heteronuclear NMR spectroscopy. , 1990, Biochemistry.

[13]  Mathias Uhlén,et al.  Fusion proteins in biotechnology and structural biology , 1992, Current Biology.

[14]  L. Kay,et al.  Simultaneous Acquisition of 15N- and 13C-Edited NOE Spectra of Proteins Dissolved in H2O , 1994 .

[15]  L. Kay,et al.  Gradient-Enhanced Triple-Resonance Three-Dimensional NMR Experiments with Improved Sensitivity , 1994 .

[16]  Richard R. Ernst,et al.  Multiple quantum filters for elucidating NMR coupling networks , 1982 .

[17]  G T Montelione,et al.  An improved strategy for determining resonance assignments for isotopically enriched proteins and its application to an engineered domain of staphylococcal protein A. , 1993, Biochemistry.

[18]  A. Bax,et al.  Empirical correlation between protein backbone conformation and C.alpha. and C.beta. 13C nuclear magnetic resonance chemical shifts , 1991 .

[19]  J. Sjödahl.  Repetitive Sequences in Protein A from Staphylococcus aureus , 1977 .

[20]  A. Braisted,et al.  Minimizing a binding domain from protein A. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[21]  A Kolinski,et al.  Folding simulations and computer redesign of protein A three‐helix bundle motifs , 1996, Proteins.

[22]  Eric Oldfield,et al.  1H, 13C and 15N chemical shift referencing in biomolecular NMR , 1995, Journal of biomolecular NMR.

[23]  S. Grzesiek,et al.  Measurement of HN-Hα J couplings in calcium-free calmodulin using new 2D and 3D water-flip-back methods , 1994, Journal of biomolecular NMR.

[24]  Gaetano T. Montelione,et al.  An efficient triple resonance experiment using carbon-13 isotropic mixing for determining sequence-specific resonance assignments of isotopically-enriched proteins , 1992 .

[25]  G. Montelione,et al.  Accurate measurements of long-range heteronuclear coupling constants from homonuclear 2D NMR spectra of isotope-enriched proteins , 1989 .

[26]  B. Nilsson,et al.  Fusions to staphylococcal protein A. , 1990, Methods in enzymology.

[27]  M. Billeter,et al.  Precise vicinal coupling constants3JHNα in proteins from nonlinear fits of J-modulated [15N,1H]-COSY experiments , 1992 .

[28]  Gaetano T. Montelione,et al.  An HCCNH Triple-Resonance Experiment Using Carbon-13 Isotropic Mixing for Correlating Backbone Amide and Side-Chain Aliphatic Resonances in Isotopically Enriched Proteins , 1993 .

[29]  J. Deisenhofer.  Crystallographic refinement and atomic models of a human Fc fragment and its complex with fragment B of protein A from Staphylococcus aureus at 2.9- and 2.8-A resolution. , 1981, Biochemistry.

[30]  J. Thornton,et al.  PROCHECK: a program to check the stereochemical quality of protein structures , 1993 .

[31]  K. Constantine,et al.  Simple Approaches for Estimating Vicinal 1H-1H Coupling-Constants and for Obtaining Stereospecific Resonance Assignments in Leucine Side Chains , 1994 .

[32]  C. Brooks,et al.  First-principles calculation of the folding free energy of a three-helix bundle protein. , 1995, Science.

[33]  M. Billeter,et al.  Calibration of the angular dependence of the amide proton-Cα proton coupling constants, 3JHNα, in a globular protein: Use of 3JHNα for identification of helical secondary structure , 1984 .

[34]  Ad Bax,et al.  Correlating Backbone Amide and Side-Chain Resonances in Larger Proteins By Multiple Relayed Triple Resonance NMR , 1992 .

[35]  R. Huber,et al.  Crystallization, crystal structure analysis and atomic model of the complex formed by a human Fc fragment and fragment B of protein A from Staphylococcus aureus. , 1978, Hoppe-Seyler's Zeitschrift fur physiologische Chemie.

[36]  R. Hodges,et al.  1H, 13C and 15N random coil NMR chemical shifts of the common amino acids. I. Investigations of nearest-neighbor effects , 1995, Journal of biomolecular NMR.

[37]  M. Llinás,et al.  1H‐15N Spin–spin couplings in alumichrome , 1978 .

[38]  Richard R. Ernst,et al.  Investigation of exchange processes by two‐dimensional NMR spectroscopy , 1979 .

[39]  G. Montelione,et al.  Phase labeling of C−H and C−C spin-system topologies: Application in PFG-HACANH and PFG-HACA(CO)NH triple-resonance experiments for determining backbone resonance assignments in proteins , 1996, Journal of biomolecular NMR.

[40]  M. Uhlén,et al.  A dual-affinity gene fusion system to express small recombinant proteins in a soluble form: expression and characterization of protein A deletion mutants. , 1990, Protein engineering.

[41]  J. Bazan,et al.  Haemopoietic receptors and helical cytokines. , 1990, Immunology today.

[42]  B. Jansson,et al.  The interaction between different domains of staphylococcal protein A and human polyclonal IgG, IgA, IgM and F(ab')2: separation of affinity from specificity. , 1993, Molecular immunology.

[43]  G. Montelione,et al.  Automated analysis of protein NMR assignments using methods from artificial intelligence. , 1997, Journal of molecular biology.

[44]  Kuo-Chen Chou,et al.  Energetic approach to the packing of α-helices. II: General treatment of nonequivalent and nonregular helices , 1984 .

[45]  B Guss,et al.  Complete sequence of the staphylococcal gene encoding protein A. A gene evolved through multiple duplications. , 1984, The Journal of biological chemistry.

[46]  G. Montelione,et al.  High-level production of uniformly 15N-and 13C-enriched fusion proteins in Escherichia coli , 1996 .

[47]  M. Uhlén,et al.  Mutational analysis of the interaction between staphylococcal protein A and human IgG1. , 1993, Protein engineering.

[48]  G. Montelione,et al.  Simulated annealing with restrained molecular dynamics using CONGEN: Energy refinement of the NMR solution structures of epidermal and type‐α transforming growth factors , 1996, Protein science : a publication of the Protein Society.

[49]  G. Montelione,et al.  The mechanism of binding staphylococcal protein A to immunoglobin G does not involve helix unwinding. , 1996, Biochemistry.

[50]  J. Wells,et al.  Comparison of a structural and a functional epitope. , 1993, Journal of molecular biology.

[51]  Richard R. Ernst,et al.  Coherence transfer by isotropic mixing: Application to proton correlation spectroscopy , 1983 .

[52]  Paul A. Keifer,et al.  Pure absorption gradient enhanced heteronuclear single quantum correlation spectroscopy with improved sensitivity , 1992 .

[53]  R. Kelley,et al.  Solution structure of the E-domain of staphylococcal protein A. , 1996, Biochemistry.

[54]  G. Montelione,et al.  Phase labeling of C−H and C−C spin-system topologies: Application in constant-time PFG-CBCA(CO)NH experiments for discriminating amino acid spin-system types , 1996, Journal of biomolecular NMR.

[55]  G. Montelione,et al.  Conformation-independent sequential NMR connections in isotope-enriched polypeptides by 1H13C15N triple-resonance experiments , 1990 .

[56]  L. Kay,et al.  Three-dimensional NOESY-HMQC spectroscopy of a 13C-labeled protein , 1990 .

[57]  H. Scheraga,et al.  Solution structure of human type-alpha transforming growth factor determined by heteronuclear NMR spectroscopy and refined by energy minimization with restraints. , 1993, Biochemistry.

[58]  M. Uhlén,et al.  A synthetic IgG-binding domain based on staphylococcal protein A. , 1987, Protein engineering.

[59]  M. Karplus,et al.  CHARMM: A program for macromolecular energy, minimization, and dynamics calculations , 1983 .

[60]  J. Richardson,et al.  Amino acid preferences for specific locations at the ends of alpha helices. , 1988, Science.

[61]  G. Rose,et al.  Helix stop signals in proteins and peptides: the capping box. , 1993, Biochemistry.

[62]  Torsion angles in the cystine bridge of oxytocin in aqueous solution. Measurements of circumjacent vicinal couplings between proton, carbon-13, and nitrogen-15 , 1980 .

[63]  P. Domaille,et al.  Four-dimensional heteronuclear triple resonance NMR methods for the assignment of backbone nuclei in proteins , 1992 .

[64]  Ad Bax,et al.  An efficient experiment for sequential backbone assignment of medium-sized isotopically enriched proteins , 1992 .

[65]  J. Sjödahl,et al.  Structural studies on the four repetitive Fc-binding regions in protein A from Staphylococcus aureus. , 1977, European journal of biochemistry.

[66]  G. Wagner,et al.  A constant-time three-dimensional triple-resonance pulse scheme to correlate intraresidue 1HN, 15N, and 13C′ chemical shifts in 15N13C-labelled proteins , 1992 .

[67]  Kuo-Chen Chou,et al.  Energetic approach to the packing of .alpha.-helixes. 1. Equivalent helixes , 1983 .

[68]  M. Karplus,et al.  Prediction of the folding of short polypeptide segments by uniform conformational sampling , 1987, Biopolymers.

[69]  G. Bodenhausen,et al.  Natural abundance nitrogen-15 NMR by enhanced heteronuclear spectroscopy , 1980 .

[70]  T F Havel,et al.  The solution structure of eglin c based on measurements of many NOEs and coupling constants and its comparison with X‐ray structures , 1992, Protein science : a publication of the Protein Society.

[71]  L. Jendeberg,et al.  Kinetic analysis of the interaction between protein a domain variants and human Fc using plasmon resonance detection , 1995, Journal of molecular recognition : JMR.

[72]  B. Borgias,et al.  MARDIGRAS : a procedure for matrix analysis of relaxation for discerning geometry of an aqueous structure , 1990 .

[73]  A Kolinski,et al.  Does a backwardly read protein sequence have a unique native state? , 1996, Protein engineering.

[74]  G. Montelione,et al.  Structures of bacterial immunoglobulin-binding domains and their complexes with immunoglobulins. , 1995, Current opinion in structural biology.