A novel ensemble-based scoring and search algorithm for protein redesign, and its application to modify the substrate specificity of the gramicidin synthetase A phenylalanine adenylation enzyme

ABSTRACT

Realization of novel molecular function requires the ability to alter molecular complex formation. Enzymatic function can be altered by changing enzyme-substrate interactions via modification of an enzyme's active site. A redesigned enzyme may either perform a novel reaction on its native substrates or its native reaction on novel substrates. A number of computational approaches have been developed to address the combinatorial nature of the protein redesign problem. These approaches typically search for the global minimum energy conformation among an exponential number of protein conformations. We present a novel algorithm for protein redesign, which combines a statistical mechanics-derived ensemble-based approach to computing the binding constant with the speed and completeness of a branch-and-bound pruning algorithm. In addition, we developed an efficient deterministic approximation algorithm, capable of approximating our scoring function to arbitrary precision. In practice, the approximation algorithm decreases the execution time of the mutation search by a factor of ten. To test our method, we examined the Phe-specific adenylation domain of the non-ribosomal peptide synthetase gramicidin synthetase A (GrsA-PheA). Ensemble scoring, using a rotameric approximation to the partition functions of the bound and unbound states for GrsA-PheA, is first used to predict binding of the wildtype protein and a previously described mutant (selective for leucine), and second, to switch the enzyme specificity toward leucine, using two novel active site sequences computationally predicted by searching through the space of possible active site mutations. The top scoring in silico mutants were created in the wetlab and dissociation / binding constants were determined by fluorescence quenching. These tested mutations exhibit the desired change in specificity from Phe to Leu. Our ensemble-based algorithm which flexibly models both protein and ligand using rotamer-based partition functions, has application in enzyme redesign, the prediction of protein-ligand binding, and computer-aided drug design.

REFERENCES

• 1: Belshaw, P., Walsh, C., and Stachelhaus, T. Aminoacyl-CoAs as probes of condensation domain selectivity in nonribosomal peptide synthesis. Science 284 (1999), 486--489.
• 2: Böhm, H. On the use of LUDI to search the fine chemicals directory for ligands of proteins of known three-dimensional structure. J. Comput. Aided Mol. Des. 8 (1994), 623--632.
• 3: Bolon, D., and Mayo, S. Enzyme-like proteins by computational design. Proc. Natl. Acad. Sci. USA 98 (2001), 14274--14279.
• 4: Bouzida, D., Rejto, P., Arthurs, S., Colson, A., Freer, S., Gehlhaar, D., Larson, V., Luty, B., Rose, P., and Verkhivker, G. Computer simulations of ligand-protein binding with ensembles of protein conformations: A monte carlo study of HIV-1 protease binding energy landscapes. Int. J. Quantum Chem. 72 (1999), 73--84.
• 5: Cane, D., Walsh, C., and Khosla, C. Harnessing the biosynthetic code:~combinations, permutations, and mutations. Science 282 (1998), 63--68.
• 6: Carlson, H. Protein flexibility and drug design:~how to hit a moving target. Curr. Opin. Chem. Biol. 6 (2002), 447--452.
• 7: Challis, G., Ravel, J., and Townsend, C. Predictive, structure-based model of amino acid recognition by nonribosomal peptide synthetase adenylation domains. Chem. Biol. 7 (2000), 211--224.
• 8: Claussen, H., Buning, C., Rarey, M., and Lengauer, T. FlexE: Efficient molecular docking considering protein structure variations. J. Mol. Biol. 308 (2001), 377--395.
• 9: Conti, E., Stachelhaus, T., Marahiel, M., and Brick, P. Structural basis for the activation of phenylalanine in the non-ribosomal biosynthesis of Gramicidin S. EMBO J. 16 (1997), 4174--4183.
• 10: Cornell, W., Cieplak, P., Bayly, C., Gould, I., Merz, K., Ferguson, D., Spellmeyer, D., Fox, T., Caldwell, J., and Kollman, P. A second generation force field for the simulation of proteins, nucleic acids and organic molecules. J. Am. Chem. Soc. 117 (1995), 5179--5197.
• 11: Decanniere, K., Transue, T., Desmyter, A., Maes, D., Muyldermans, S., and Wyns, L. Degenerate interfaces in antigen-antibody complexes. J. Mol. Biol. 313 (2001), 473--478.
• 12: Desmet, J., Maeyer, M., Hazes, B., and Lasters, I. The dead-end elimination theorem and its use in protein side-chain positioning. Nature 356 (1992), 539--542.
• 13: Doekel, S., and Marahiel, M. Dipeptide formation on engineered hybrid peptide synthetases. Chem. Biol. 7 (2000), 373--384.
• 14: Ehmann, D., Trauger, J., Stachelhaus, T., and Walsh, C. Aminoacyl-SNACs as small-molecule substrates for the condensation domains of nonribosomal peptide synthetases. Chem. Biol. 7 (2000), 765--772.
• 15: Eisenberg, D., and McLachlan, A. Solvation energy in protein folding and binding. Nature 319 (1984), 199--203.
• 16: Eppelmann, K., Stachelhaus, T., and Marahiel, M. Exploitation of the selectivity-conferring code of nonribosomal peptide synthetases for the rational design of novel peptide antibiotics. Biochemistry 41 (2002), 9718--9726.
• 17: Fauchére, J., and Pliška, V. Hydrophobic parameters Φ of amino-acid side chains from the partitioning of n-acetyl-amino-acid amides. Eur. J. Med. Chem. Chim. Ther. 18 (1983), 369--375.
• 18: Hellinga, H., and Richards, F. Construction of new ligand binding sites in proteins of known structure:~I. Computer-aided modeling of sites with pre-defined geometry. J. Mol. Biol. 222 (1991), 763--785.
• 19: Hill, T. Statistical Mechanics: Principles and Selected Applications. McGraw-Hill Book Company, Inc., New York., New York, 1956, ch. 1, "Principles of Classical Statistical Mechanics", pp. 1--17.
• 20: Jaramillo, A., Wernisch, L., Héry, S., and Wodak, S. Automatic procedures for protein design. Comb. Chem. High Throughput Screen. 4 (2001), 643--659.
• 21: Jin, W., Kambara, O., Sasakawa, H., Tamura, A., and Takada, S. De novo design of foldable proteins with smooth folding funnel: Automated negative design and experimental verification. Structure 11 (2003), 581--591.
• 22: Keating, A., Malashkevich, V., Tidor, B., and Kim, P. Side-chain repacking calculations for predicting structures and stabilities of heterodimeric coiled coils. Proc. Natl. Acad. Sci. USA 98 (2001), 14825--14830.
• 23: Knegtel, R., Kuntz, I., and Oshiro, C. Molecular docking to ensembles of protein structures. J. Mol. Biol 266 (1997), 424--440.
• 24: Kuntz, I., Blaney, J., Oatley, S., Langridge, R., and Ferrin, T. A geometric approach to macromolecular-ligand interactios. J. Mol. Biol. 161 (1982), 269--288.
• 25: Lasters, I., and Desmet, J. The fuzzy-end elimination theorem:~correctly implementing the side chain placement algorithm based on the dead-end elimination theorem. Protein Eng. 6 (1993), 717--722.
• 26: Lilien, R., Sridharan, M., Huang, J., Bushweller, J., and Donald, B. Computational screening studies for Core Binding Factor Beta: Use of multiple conformations to model receptor flexibility. Poster - 8th International Conference on Intelligent Systems for Molecular Biology ISMB-2000.
• 27: Linne, U., Doekel, S., and Marahiel, M. Portability of epimerization domain and role of peptidyl carrier protein on epimerization activity in nonribosomal peptide synthetases. Biochemistry 40 (2001), 15824--15834.
• 28: Looger, L., Dwyer, M., Smith, J., and Hellinga, H. Computational design of receptor and sensor proteins with novel functions. Nature 423 (2003), 185--190.
• 29: Lorber, D., and Shoichet, B. Flexible ligand docking using conformational ensembles. Protein Sci. 7 (1998), 938--950.
• 30: Lovell, S., Word, J., Richardson, J., and Richardson, D. The penultimate rotamer library. Proteins 40 (2000), 389--408.
• 31: Marvin, J., and Hellinga, H. Conversion of a maltose receptor into a zinc biosensor by computational design. PNAS 98 (2001), 4955--4960.
• 32: McQuarrie, D. Statistical Mechanics. Harper & Row, New York, 1976.
• 33: Montfort, W., Perry, K., Fauman, E., Finer-Moore, J., Maley, G., Hardy, L., Maley, F., and Stroud, R. Structure, multiple site binding, and segmental accommodation in thymidylate synthase on binding dUMP and an anti-folate. Biochemistry 29 (1990), 6964--6977.
• 34: Mootz, H., Schwarzer, D., and Marahiel, M. Construction of hybrid peptide synthetases by module and domain fusions. Proc. Natl. Acad. Sci. USA 97 (2000), 5848--5853.
• 35: Murthy, K., Winborne, E., Minnich, M., Culp, J., and Debouck, C. The Crystal Strucutres at 2.2-Å Resolution of Hydroxyethylene-based Inhibitors Bound to Human Immunodeficiency Virus Type 1 Protease Show the Inhibitors Are Present in Two Distinct Orientations. J. Biol. Chem. 267 (1992), 22770--22778.
• 36: Offredi, F., Dubail, F., Kischel, P., Sarinski, K., Stern, A., Van de Weerdt, C., Hoch, J., Prosperi, C., Fran\ccois, J., Mayo, S., and Martial, J. De novo backbone and sequence design of an idealized α/β-barrel protein:~evidence of stable tertiary structure. J. Mol. Biol. 325 (2003), 163--174.
• 37: Österberg, F., Morris, G., Sanner, M., Olson, A., and Goodsell, D. Automated docking to multiple target structures: Incorporation of protein mobility and structural water heterogeneity in autodock. Proteins 46 (2002), 34--40.
• 38: Philippopoulos, M., and Lim, C. Exploring the dynamic information content of a protein NMR structure: Comparison of a molecular dynamics simulation with the NMR and X-Ray structures of Escherichia coli ribonuclease HI. Proteins 36 (1999), 87--110.
• 39: Pierce, N., and E., W. Protein design is NP-hard. Protein Eng. 15 (2002), 779--782.
• 40: Pierce, N., Spriet, J., Desmet, J., and Mayo, S. Conformational splitting:~a more powerful criterion for dead-end elimination. J. Comput. Chem. 21 (2000), 999--1009.
• 41: Ponder, J., and Richards, F. Tertiary templates for proteins:~use of packing criteria in the enumeration of allowed sequences for different structural classes. J. Mol. Biol. 193 (1987), 775--791.
• 42: Raag, R., and Poulos, L. Crystal structures of cytochrome P-450CAM complexed with camphane, thiocamphor, and adamantane: factors controlling P-450 substrate hydroxylation. Biochemistry 30 (1991), 2674--2684.
• 43: Rienstra, C., Tucker-Kellogg, L., Jaroniec, C., Hohwy, M., Reif, B., McMahon, M., Tidor, B., Lozano-Pérez, T., and Griffin, R. De novo determination of peptide structure with solid-state magic-angle spinning NMR spectroscopy. Proc. Natl. Acad. Sci. USA 99 (2002), 10260--10265.
• 44: Samanta, U., Pal, D., and Chakrabarti, P. Packing of aromatic rings against tryptophan residues in proteins. Acta Cryst. D 55 (1999), 1421--1427.
• 45: Schneider, A., Stachelhaus, T., and Marahiel, M. Targeted alteration of the substrate specificity of peptide synthetases by rational module swapping. Mol. Gen. Genet. 257 (1998), 308--318.
• 46: Schwarzer, D., Finking, R., and Marahiel, M. Nonribosomal peptides:~from genes to products. Nat. Prod. Rep. 20 (2003), 275--287.
• 47: Shifman, J., and Mayo, S. Modulating calmodulin binding specificity through computational protein design. J. Mol. Biol. 323 (2002), 417--423.
• 48: Stachelhaus, T., and Marahiel, M. Modular structure of peptide synthetases revealed by dissection of the multifunctional enzyme GrsA. J. Biol. Chem. 270 (1995), 6163--6169.
• 49: Stachelhaus, T., Mootz, H., and Marahiel, M. The specificiy-conferring code of adenylation domains in nonribosomal peptide synthetases. Chem. Biol. 6 (1999), 493--505.
• 50: Stachelhaus, T., Schneider, A., and Marahiel, M. Rational design of peptide antibiotics by targeted replacement of bacterial and fungal domains. Science 269 (1995), 69--72.
• 51: Street, A., and Mayo, S. Computational protein design. Structure 7 (1999), R105--R109.
• 52: Lisa Tucker-Kellogg , Tomas Lozano-Perez, Systematic conformational search with constraint satisfaction, 2002
• 53: Weber, T., Baumgartner, R., Renner, C., Marahiel, M., and Holak, T. Solution structure of PCP, a prototype for the peptidyl carrier domains of modular peptide synthetases. Structure 8 (2000), 407--418.
• 54: Weiner, S., Kollman, P., Case, D., Singh, U., Ghio, C., Alagona, G., Profeta, S., and Weiner, P. A new force field for molecular mechanical simulation of nucleic acids and proteins. J. Am. Chem. Soc. 106 (1984), 765--784.
• 55: Wojtczak, J., Cody, V., Luft, J., and Pangborn, W. Structures of human transthyretin complexed with thyroxine at 2.0Å resolution and 3',5'-Dinitro-N-acetyl-L-thyronine at 2.2Å resolution. Acta Cryst. D 52 (1996), 758--765.
• 56: Zhou, G., Ferrer, M., Chopra, R., Kapoor, T., Strassmaier, T., Weissenhorn, W., Skehel, J., Oprian, D., Schreiber, S., Harrison, S., and Wiley, D. The structure of an HIV-1 specific cell entry inhibitor in complex with the HIV-1 gp41 trimeric core. Bioorg. Med. Chem. 8 (2000), 2219--2228.

INDEX TERMS

Primary Classification:
  J. Computer Applications
  J.3 LIFE AND MEDICAL SCIENCES
      Subjects: Biology and genetics

General Terms:
Algorithms, Design, Experimentation, Measurement

Keywords:
enzyme design, fluorescence binding assay, molecular ensemble, non-ribosomal peptide synthetase, protein design, protein flexibility, protein-ligand binding