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AddressDepartment of Biochemistry, University of Cambridge, 80 Tennis CourtRoad, Cambridge CB2 1GA, UK

Current Opinion in Structural Biology 2000, 10:399–400

In the past few years, much effort has been directed towardsthe problem of how to create proteins with tailored functionscapable of performing specific roles in user-defined environ-ments. Exhaustive screening of microbes has the potential totap into pre-evolved diversity, but this serendipitous approachmay be limited in its ability to uncover naturally occurring pro-teins capable of functioning in non-natural conditions.Mainstream attention, therefore, remains largely focused ontwo fundamentally different ways in which the creation toorder of new biological macromolecules can be achieved bydirect engineering of protein function. The first involves denovo design of a biomolecule that folds in solution in such away as to bind a substrate and, in the case of an enzyme, to pre-sent appropriate catalytic functional groups to that substrate.The second involves rational, rather than true, design andrequires a system for selecting from a large pool of randomlygenerated biomolecules the few that present an effective arrayof catalytic functional groups to a substrate. Rational redesignof an existing protein falls somewhere between these twoapproaches. This section aims to review the current state ofthe art in these different areas of protein design.

Creation of novel proteins by de novo design requires a detailedunderstanding of, and control over, biomolecular conformationand reactivity. It is potentially the most intellectually satisfyingsolution to engineering protein function, yet it is a formidableproblem, requiring answers to fundamental questions such as‘what is the rate-limiting transformation in the reactionsequence’, ‘where must functional groups be placed in spacerelative to the substrates to catalyse the transformation’, ‘whatcavity will differentially bind the substrate, transition states,intermediates and products’ and ‘what polypeptide will foldcorrectly to deliver all this functionality’? The first two ques-tions require nontrivial theoretical calculations on reactiondynamics; this is increasingly possible now, but is realisticallybeyond the scope of this section. The folding question hasbeen reviewed extensively elsewhere recently and it is nowclear that substantial progress is being made in the design offour-helix bundles, with improvements being seen particularlyin the packing of hydrophobic cores. Beyond this, the moststriking example to date is probably the de novo design of a pro-tein that folds into a compact ββα structure. The next step thenis presumably to try to circumvent the size limitations imposedby the available computational power, such that larger, morecomplex structures can be designed. However, these examplesonly strictly address the inverse folding question of ‘whatsequence is compatible with this fold?’, whilst answers to thetrue folding question of ‘what 3D shape will a given

polypeptide adopt?’ remain elusive. For the time being then, itseems that de novo design of an enzyme will require a prioriknowledge of which protein fold is compatible with the desiredfunctionality, in itself not a simple question!

The question of how to design a protein cavity to bind a givenligand seems to have received less attention to date. It is, how-ever, essentially the same type of problem, albeit potentiallymore difficult, as how to design a ligand for a given protein cav-ity. Gane and Dean (pp 401–404) provide a comprehensivereview of recent literature in the area of rational drug design.They discuss potential applications of structure-based drugdesign, including in silico high-throughput screening (HTS)programs capable of accessing a much greater diversity ofchemical structures than is experimentally available, whilst pre-dicting the mode of binding of any experimental HTS hits toallow rational improvements to be made in a hits-to-leads pro-gram. This latter aspect would seem to be in direct competitionwith high-throughput protein–ligand crystallography and it willbe interesting to see which proves to be the more reliable in thenext few years; neither approach, of course, addresses the issueof predicting the selectivity of the lead compounds and novelapproaches to this are still needed. The current crop of algo-rithms discussed in this review seem very much enthalpyorientated and are heavily restricted in their ability to deal withentropic factors such as solvation/desolvation. It is also unclearwhether they can deal with significant reorganisation of theprotein structure on ligand binding, yet it seems reasonable tosuppose that such factors will be important in any protein–lig-and binding event, irrespective of whether you are lookingfrom the protein or the ligand perspective. Not surprisinglythen, the number of drug candidates designed entirely in silicois still small. More pertinent to this section is the inference thatthe converse problem of designing a cavity capable of reorgan-ising more or less subtly both on ligand binding andsubsequently throughout the reaction coordinate is clearly amammoth task that has yet to be achieved.

One approach that aims to circumvent many of the problemsidentified above, including prediction of folding and design ofligand-binding pockets, is rational redesign of proteins.Quéméneur et al. (pp 405–410) discuss recent examples inenzyme redesign in which substrate specificity has beenchanged by rational site-directed mutation of binding pocketswhilst keeping the same catalytic mechanism, or in which cat-alytic mechanisms have been re-engineered by judiciousintroduction of catalytic residues without perturbing the bindingspecificity. The mutation of cyclophilin to create a proline-spe-cific endopeptidase by introducing a serine-protease-likecatalytic triad is perhaps the most spectacular success yet of ratio-nal redesign, but it is important not to get too carried awaybecause things might not always be what they seem. In serineproteases, mutation of the key histidine disrupts the catalytic

Engineering and design: Rational versus combinatorial approachesEditorial overviewJonathan Blackburn

triad and reduces the rate enhancement by 106-fold; the engi-neered peptidase shows impressive rate enhancements of>1011-fold, but mutation of both the equivalent histidine and theaspartate only reduces the rate enhancement by 20-fold. Thisperhaps suggests that the origin of the rate enhancement in thedesigned protein is less to do with the catalytic triad than it is todo with the original function of cyclophilin and the positioning ofthe introduced serine; in the course of a cis-trans proline isomeri-sation, the prolyl peptide bond will necessarily at least transientlypass through a conformation in which resonance stabilisation ofthe amide linkage has been disrupted, potentially rendering thecarbonyl moiety more ketone-like in its reactivity. A suitablypositioned nucleophile, such as the β-hydroxyl of a serineresidue, might then simply intercept this reactive state and divertthe reaction coordinate towards bond cleavage. Thus, in general,merely grafting binding or catalytic machineries onto other exist-ing scaffolds may not be sufficient to promote efficient bindingor catalysis and other mutations introduced at random may alsobe necessary to effect the desired function. The review com-ments that many proteins in a superfamily catalyse seeminglydiverse chemical transformations, yet apparently conserve cer-tain specific chemical properties. The cited example of theenolase family could potentially be broadened by noting the rel-ative prevalence of enediol intermediates in reactions catalysedby α/β-barrel proteins; observations of this type might prove use-ful for future rational redesign experiments, as they perhapssuggest that certain superfamilies are intrinsically better atcatalysing particular reaction types than others.

Choo and Isalan (pp 411–416) look in more detail at the redesignof zinc finger proteins, highlighting progress towards the genera-tion of a zinc finger ‘tool box’ from which proteins capable ofrecognising any defined sequence can be built and discussingthe role of synergistic interactions between adjacent fingers indetermining binding specificity. It will be interesting to seewhether similar factors also have a role to play elsewhere inattempts to redesign other apparently modular systems. Theyalso observe that specific recognition of extended DNAsequences can be achieved via dimerisation of zinc finger pairs;this, together with recent reports of the chemical induction ofdimerisation, raises the tantalising prospect of designed tran-scription factors whose binding to a defined DNA sequence iscontrolled by the administration of a synthetic organic molecule.Such combinations might have potential in gene therapy appli-cations or developmental biology for the conditional(de)activation of specific genes. McCafferty and Glover(pp 417–420) discuss the importance of understanding why ther-apeutic proteins fail and illustrate how this has been applied togenerate viable therapeutic ‘humanised’ antibodies withimproved pharmacokinetic and reduced immunogenic proper-ties. This work has clearly benefited significantly from access tostructural data through accurately defining domain or loopboundaries. Simply grafting CDRs from murine to human anti-body scaffolds often results, however, in loss of affinity, requiringadditional second-sphere changes to restore activity; these wouldhave been difficult to predict from structure, neatly demonstrat-ing the advantages of combining selection and knowledge-baseddesign processes in generating therapeutic proteins.

The work discussed on rational redesign of function all relies toa greater or lesser extent on the premise that structural data isinvaluable in guiding the mutations introduced. Tobin et al.(pp 421–428), however, argue that our ability to predict theeffects of multiple mutations (or even to rationalise their effectswith hindsight) is poor at present and that a Darwinian schemeof iterative rounds of random mutation, recombination andselection is the better way forward, with structural data nolonger a prerequisite. This view is supported by the fascinatingresults of family shuffling experiments, in which the best-evolved clones are often so far removed from the parents thatthey would not have been predicted from structure. The impli-cation that genotypic traits in parents are not always manifestedphenotypically leads to difficulties in rationalising geneticpotential to evolve, as the best-evolved clone will not necessar-ily be closest in sequence to the best parent. By extension, ofcourse, this suggests that even directed evolution experimentswill be fraught with difficulty if the wrong starting point is cho-sen! Random mutation and recombination of preselecteddiversity seems well suited to engineering multiple existingtraits, such as thermal stability or solvent stability. However, if anentirely new trait is sought, implicit compatibilities with theoriginal function may yet impose unnecessary constraints on theability of the system to evolve. The review also discusses thepossibility of putting together individual intact functional motifsvia recombination to generate new function; modular proteins,such as polyketide synthases and nonribosomal peptide syn-thases, represent obvious targets, but another enticingpossibility is that smaller, stable subdomains of individual pro-teins, such as the phosphate-binding motif found conserved insome α/β-barrel proteins, might be recombined to generateentirely new proteins exhibiting different functions. With suchnew approaches in the pipeline, the future of directed evolutionappears bright, although the ability to engineer proteins willdoubtless rely heavily on the quality of the selections or screensdevised to find the needle in the haystack.

Lowe (pp 428–434) changes tack from engineering proteinsper se to the design, fabrication and utility of nanostructuresas biomimetic analogues of wires, motors and informationstorage/retrieval systems, etc. If this sounds like science fic-tion, read on to discover that it is not! The review concludesthat nanobiology has much to offer in demonstrating how torecognise, organise, functionalise and assemble new molecular materials.

This section shows that both structure-based redesign anddirected evolution are capable of engineering proteins withpharmaceutically, chemically or medically useful properties; itis likely that this will continue to be the case. It seems reason-able to suppose though that a judicious marriage of these twoapproaches might ultimately prove the most successful route ofall and it is interesting in this regard that a combined methodhas recently been used to create a man-made protein that notonly mimics, but actually surpasses nature’s own catalyticdesign. The Holy Grail, how to create to order any tailor-madebiocatalyst, still remains to be found, but we seem to be gettinga much better idea of how to use the tools available to us now!

400 Engineering and design