PERSPECTIVES 70. van Horck, F. P., Ahmadian, M. R., Haeusler, L. C., membrane traffic along microtubules in living cells. J. Cell coverage of this subject. The examples chosen Moolenaar, W. H. & Kranenburg, O. Sci. 112, 21–33 (1999). Characterization of p190RhoGEF, a RhoA-specific 78. Bershadsky, A. D. & Futerman, A. H. therefore reflect our own experiences and guanine nucleotide exchange factor that Disruption of the Golgi apparatus by brefeldin perspectives, and many significant topics and interacts with microtubules. J. Biol. Chem. 276, A blocks cell polarization and inhibits directed cell 4948–4956 (2001). migration. Proc. Natl Acad. Sci. USA 91, 5686–5689 achievements have necessarily been omitted 71. Ren, X. D., Kiosses, W. B. & Schwartz, M. A. Regulation (1994). or abbreviated. of the small GTP-binding protein Rho by cell 79. Rodionov, V. I. et al. Microtubule-dependent adhesion and the cytoskeleton. EMBO J. 18, 578–585 control of cell shape and pseudopodial (1999). activity is inhibited by the antibody to Kicking off with TyrRS 72. Fukata, Y., Amano, M. & Kaibuchi, K. Rho–Rho-kinase kinesin motor domain. J. Cell Biol. 123, 1811–1820 pathway in smooth muscle contraction and cytoskeletal (1993). TyrRS proved to be a fruitful system for the reorganization of non-muscle cells. Trends Pharmacol. dissection of enzyme catalysis by site-directed Sci. 22, 32–39 (2001). Acknowledgements 73. Bershadsky, A. D., Vaisberg, E. A. & Vasiliev, J. M. We thank O. Krylyskina and G. Resch for their invaluable help with mutagenesis. It is a central enzyme in molecu- Pseudopodial activity at the active edge of migrating the videos and animation. A.B. and B.G. are grateful to D. Riveline lar biology and is responsible for ligating the fibroblast is decreased after drug-induced microtubule and J. Kirchner for providing the experimental data for figures 2 depolymerization. Cell Motil. Cytoskeleton 19, 152–158 and 3. A. Huttenlocher is acknowledged for providing the DsRed amino-acid tyrosine (Tyr) to its cognate (1991). zyxin construct that was used in figure 5. This work was sup- tRNATy r in an ATP-dependent reaction that 74. Dunn, G. A., Zicha, D. & Fraylich, P. E. Rapid, ported in part by a grant from the Austrian Science Research Ty r microtubule-dependent fluctuations of the cell margin. Council to J.V.S. and I.K. B.G. is the incumbent of the E. Neter produces tyrosyl–tRNA . As with all J. Cell Sci. 110, 3091–3098 (1997). Chair in Cell and Tumor Biology; A.B. holds the J. Moss Chair of aminoacyl–tRNA synthetases, the accurate 75. Waterman-Storer, C. M., Worthylake, R. A., Liu, B. P., Biomedical Research. Burridge, K. & Salmon, E. D. Microtubule selection of the cognate amino acid is impor- growth activates Rac1 to promote lamellipodial tant for the faithful translation of the genetic protrusion in fibroblasts. Nature Cell Biol. Online links 1, 45–50 (1999). code. In particular, in the living cell, TyrRS 76. Bretscher, M. S. & Aguado-Velasco, C. Membrane traffic DATABASES discriminates against the most closely related during cell locomotion. Curr. Opin. Cell Biol. 10, 537–541 The following terms in this article are linked online to: (1998). Swiss-Prot: http://www.expasy.ch/ amino acid to tyrosine — phenylalanine 77. Toomre, D., Keller, P., White, J., Olivo, J. C. & Simons, K. Cdc42 | Dia1 | Rho kinase (Phe) — which lacks only the phenolic Dual-color visualization of trans-Golgi network to plasma Access to this interactive links box is free online. hydroxyl group of tyrosine. The Bacillus stearothermophilus TyrRS was ripe for engineering. Greg Winter’s labora- tory had determined the sequence of the enzyme by using a combination of classical TIMELINE protein sequencing and DNA sequencing of the cloned gene, and this gene had also been expressed in Escherichia coli. David Blow’s Protein engineering 20 years on laboratory had crystallized the enzyme and was in the final stages of solving its structure. James A. Brannigan and Anthony J. Wilkinson Mechanistic studies of the enzyme in Alan Fersht’s laboratory had shown the existence It is 20 years since site-directed modify these functional groups specifically of a remarkably stable aminoacyl-adenylate mutagenesis was first used to modify the and to explore the effects on activity. This intermediate. This meant that the enzyme active site of an enzyme of known structure could only be achieved by painstaking chem- and mechanism. Since then, this method ical modification with its attendant prob- His45 has contributed far-reaching insights into lems of limited range and poor specificity. In Thr40 O N catalysis, specificity, stability and folding of this climate, the arrival of site-directed OH O H N O proteins. Engineered proteins are now being mutagenesis, a technique that allowed O N His48 O N used in industry and for the improved amino-acid sequences in proteins to be OO H N O O Asp78 P N treatment of human disease. altered at will, was the answer to an enzy- O O N N mologist’s prayer. The first uses of this tech- O O O HH O N O N H At the beginning of the 1980s, a major stum- nique, to mutate genes that encode enzymes H O H HNH O bling block to progress in biochemistry was of known mechanism and produce proteins Tyr169 O O H Thr51 our inability to direct chemistry specifically with defined amino-acid residue substitu- S O at macromolecular surfaces in a way that tions, were reported for tyrosyl–transfer O H Gln195 Cys35 O allowed the relationship between structure RNA synthetase (TyrRS) and β-lactamase at H O Tyr34 and activity to be examined in detail. the end of 1982 (REFS 4–6). Asp176 Nowhere was this limitation more acutely These precise changes of only one or two felt than in the field of enzymology. The amino-acid residues were later followed by Figure 1 | The active site of tyrosyl–transfer 7 8 RNA synthetase. The modelled structure of the principles that govern enzyme catalysis were changes of entire loops and even domains . transition state in tyrosyl-adenylate formation is 1,2 understood and the number of enzyme This construction of modified proteins and shown. The transition state was extrapolated from structures solved by X-ray crystallography the analysis of their properties coalesced to the known structure of the enzyme-bound tyrosyl- was beginning to grow, albeit slowly3. These form a new field — that of protein engineer- adenylate. Hydrogen-bonding interactions structures, with their stereochemical clarity, ing. In this article, we have traced the field of between the enzyme and transition state species provided a framework for formulating protein engineering over the past 20 years, are shown as dashed lines. The roles of threonine (Thr) 40 and histidine (His) 45 are discussed in the mechanisms of action in which precise roles principally following the thread of enzyme main text. Asp, aspartic acid; Cys, cysteine; Gln, were attributed to functional groups that engineering that was pioneered in those early glutamine; Tyr, tyrosine. Modified with permission were pinpointed in the active sites. The nat- papers (TIMELINE). It is not possible in an arti- from REF. 10 © (2002) National Academy of ural way to test emerging hypotheses was to cle of this length to provide comprehensive Sciences, USA. 964 | DECEMBER 2002 | VOLUME 3 www.nature.com/reviews/molcellbio PERSPECTIVES Timeline | Twenty years of protein engineering First mutations of proteins of Hydrogen bonding Directed evolution of First chimeric Design of a Lysozyme known contributions Monomeric insulins Phage display a protease active in antibody approved protease active at mechanism mechanism4–6. quantitated10. designed74. of antibodies88. organic solvents46. for clinical use. 100 °C (REF. 28). revisited20. 1978 1982 1984 1985 1986 1988 1989 1990 1992 1993 1994 1995 1996 1998 1999 2001 First use of Protein engineering CDR grafting in Protein-folding Designed First uses of the Engineered First use of a tailored site-directed of improved activity antibodies7. pathway haemoglobin for a gene-shuffling insulins reach enzyme in a crop mutagenesis80. in TyrRS9. described22. blood substitute68. approach48. the market. plant58. CDR, complementarity-determining regions; TyrRS, tyrosyl–tRNA synthetase. could be assayed by active-site titration, and it was also possible to show that the extra bonding contributions in TyrRS12 indicated therefore accurate and reproducible steady- binding energy (~1 kcal mol–1) provided by that deletions that leave an unpaired, state kinetic measurements of reaction rates the Cys35 side chain in the wild-type enzyme uncharged hydrogen-bond donor or accep- could be made. It also allowed the crystallog- is used to stabilize the bound substrate in its tor weaken binding by ~1 kcal mol–1,but raphers to grow crystals and determine a transition state rather than its ground state. deletions that leave an unpaired, charged structure of the enzyme in its complex with This use of binding energy selectively in group weaken binding by ~4 kcal mol–1.The tyrosyl-adenylate, which showed that there transition-state stabilization was shown in a avoidance of unsolvated charge is therefore were a plethora of contacts between the dramatic way when modelling and mutagen- an important component of biological speci- enzyme and the intermediate, including a esis were used to probe the contributions to ficity, which accounts for the presence of dozen or so hydrogen bonds (FIG. 1). This catalysis of threonine 40 (Thr40) and histi- aspartate 176 (Asp176) at the base of the detailed understanding of the structure and dine 45 (His45)10. These residues are far away amino-acid side-chain binding pocket in mechanism of the enzyme meant that site- from the seat of the reaction (FIG. 1),but TyrRS (FIG. 1). A Tyr ligand with its phenolic directed mutagenesis could be used to together they contribute 300,000-fold to the hydroxyl group can form a charge-dipole address specific well-formulated questions.