Structure and Evolutionary Analysis of a Non-biological ... · Structure and Evolutionary Analysis of a Non-biological ATP-binding Protein Sheref S. Mansy1, Jinglei Zhang2, Rainer

doi:10.1016/j.jmb.2007.05.062 J. Mol. Biol. (2007) 371, 501–513

Structure and Evolutionary Analysis of aNon-biological ATP-binding Protein

Sheref S. Mansy1, Jinglei Zhang2, Rainer Kümmerle3, Mikael Nilsson2

James J. Chou4, Jack W. Szostak1 and John C. Chaput2⁎

1Howard Hughes MedicalInstitute, and Departmentof Molecular Biology,Massachusetts GeneralHospital, Boston, MA 02114,USA2Center for BioOpticalNanotechnology, The BiodesignInstitute, and Department ofChemistry and Biochemistry,Arizona State University,Tempe, AZ 85287-1604, USA3Bruker BioSpin AG,Fällanden, Switzerland4Department of BiologicalChemistry and MolecularPharmacology, HarvardMedical School, Boston,MA 02115, USA

Abbreviations used: NOE, nucleaenhancement; MBP, maltose bindingbinding protein; RCD, residual dipofolding optimized family B.E-mail address of the correspondi

[email protected]

0022-2836/$ - see front matter © 2007 E

We present a structural and functional analysis of the evolutionary op-timization of a non-biological protein derived from a library of randomamino acid sequences. A series of previously described in vitro selectionexperiments transformed a low-affinity ancestral sequence into a stablyfolded, high affinity ATP binding protein structure. While the evolutionarilyoptimized protein differs from its ancestral sequence through the accu-mulation of 12 amino acid mutations, the means by which those mutationsenhance the stability and functionality of the protein were not wellunderstood. We used a combination of mutagenesis, biochemistry, andNMR spectroscopy to investigate the structural and functional significanceof each mutation. We solved the three-dimensional structure of the foldingoptimized protein by solution NMR, which revealed a fourth strand of theβ-sheet of the α/β-fold that was not observed in an earlier crystallographicanalysis of a less stable version of the protein. The structural rigidity of thenewly identified β-strand was confirmed by T1, T2, and heteronuclearnuclear Overhauser enhancement (NOE) measurements. Biochemicalexperiments were used to examine point mutations that revert theoptimized protein back to the ancestral residue at each of the 12 sites. Acombination of structural and functional data was then used to interpret thesignificance of each amino acid mutation. The enhanced ATP affinity waslargely due to the emergence of a patch of positive charge density on theprotein surface, while the increased solubility resulted from severalmutations that increased the hydrophilicity of the protein surface, therebydecreasing protein aggregation. One mutation may stabilize the hydro-phobic face of the β-sheet.

© 2007 Elsevier Ltd. All rights reserved.

*Corresponding author
Keywords: mRNA display; solution NMR; in vitro evolution
Introduction

We previously used mRNA display1 to isolate anon-biological ATP-binding protein from anunbiased random-sequence library.2 The libraryfrom which our ancestral ATP-binding proteinwas isolated consisted of N1012 unique proteinseach containing a central random region of 80 resi-

r Overhauserprotein; ABP, ATPlar coupling; FOB,

ng author:

lsevier Ltd. All rights reserve

dues. After eight rounds of mRNA display based invitro selection and amplification, four families ofATP binding proteins were identified from thestarting library. None of these four familiesexhibited any significant sequence homology toany other known amino acid sequence found innature. One of these protein families, family B, wasfurther optimized for improved binding activitythrough mutagenesis and several additional roundsof selection and amplification.2 Analysis of theprotein sequences from this selection revealed twoconserved CXXC motifs that bound a single zinc-metal ion. Complete biophysical characterization ofthe binding optimized proteins from the output ofthis selection was complicated by the presence ofadditional amino acid sequences outside the core-binding domain of the selected ATP-binding protein.

d.

mailto:[email protected]

502 Analysis of a Non-biological ATP-binding Protein

Identification of the core-binding domain by thecharacterization of truncation constructs allowed forthe in vivo expression and isolation of monomericprotein in pure form. The X-ray crystal structure ofone highly divergent variant (protein 18–19) fromthis selection was reported3 and reveals a novel α/βfold not found in biology with a metal binding siteremarkably similar to the treble clef zinc bindingmotif.4

All of the variants examined required highconcentrations of free ATP in order to remainstably folded and soluble. In an attempt to over-come this limitation, mRNA display was used toselect for new protein variants that remained stablyfolded in the absence of excess ATP ligand.5 Theselection strategy involved using guanidine hydro-chloride to enrich for protein variants thatremained folded, and therefore functional, in thepresence of increasing concentrations of chemicaldenaturant. For each round of selection, theguanidine hydrochloride concentration was gradu-ally increased to ensure that less than 10% of theinput into each round of selection was recoveredfrom the previous round. After six rounds ofselection, many of these folding optimized ATPbinding proteins remained water soluble andmonomeric when expressed in Escherichia coli, andbound ATP with high affinity and specificity. Wewere then able to identify a 62 amino acid residuecore domain of this protein that retained the sameATP binding function as the full-length construct.Comparison of the consensus sequences for eachstage in the evolution of this protein (Figure 1(a)),from ancestral sequence to the folding optimized

Figure 1. Sequence alignment of ATP binding protein vaoptimized, and folding-optimized consensus sequences fromof our denaturing selection,5 respectively, and protein 18–1mutant proteins used to identify genetic mutations with strselected mutation was reverted back to its ancestral residue.mutations are shown in bold. Apparent solution bindingequilibrium filtration.14

variant, revealed the location and identity of 12amino acid substitutions that arose during the invitro evolution of this protein from a low affinityancestral state to a well folded high affinitystructure.We used structural and functional studies to

investigate this in vitro evolutionary processes ingreater detail. We solved the tertiary structure ofthe core ATP binding domain of the foldingoptimized protein by nuclear magnetic resonancespectroscopy. Analysis of the folded structurerevealed a fourth strand in the β-sheet that wasnot present in the crystal structure of a less stableand highly divergent variant. The rigidity of thefourth β-strand was confirmed by T1, T2, andheteronuclear nuclear Overhauser enhancement(NOE) measurements. To investigate the functionalproperties of the individual mutations, we con-structed single-point revertants for each mutationpresent in the core-binding domain of the proteinand evaluated each revertant for its affect on ATPbinding affinity, protein solubility, and thermalstability. We then used the structure of the foldingoptimized protein to interpret the effect of eachmutation on ATP affinity and protein solubility.Taken together, the data suggest that a poorlyfunctional, artificial protein can be significantlyoptimized through the accumulation of single-pointmutations that do not dramatically affect the fold ofthe protein or the mode of ligand binding. Thissuggests that it might be possible to evolve manybiologically relevant proteins that are structurallyunstable or poorly soluble to a state of increasedfolding stability.

riants of family B. (a) Alignment of primordial, binding-rounds 8 and 18 of our previous selection2 and round 69. (b) Amino acid sequences of individual single-pointuctural and/or functional significance. In each case, theInvariant residues are indicated by dashes and selectedaffinity for ATP (Kd) was determined by displacement

503Analysis of a Non-biological ATP-binding Protein

Results

Effects of mutations on ATP-binding

Our initial goal was to follow changes in ATP-affinity during the evolutionary optimization of theancestral ATP-binding protein and to determine theeffects of the individual mutations on ATP affinity.We therefore prepared the ancestral, binding opti-mized, folding optimized, and single-mutant rever-tant proteins as maltose binding protein (MBP)fusion constructs, since most of the proteins inves-tigated were not soluble as autonomous foldingunits. Each ATP binding protein was constructed inthe 62 amino acid sequence context defined by thecore-binding domain of the folding optimizedvariant. Comparison of the dissociation constantsfor the ancestral, binding optimized, and foldingoptimized proteins (Figure 1(a)) revealed a dramaticshift in ATP binding. The Kd value of the ancestralprotein for ATPwas estimated to be between 0.5 mMand 1 mM, while the binding optimized protein hada Kd value of 100 μM. Remarkably, the Kd of thefolding optimized protein decreased dramatically to∼500 nM, suggesting that selection for greaterstability of the folded state had indirectly led to anincrease in ATP-affinity.To evaluate the significance of each of the

mutations that arose during the in vitro evolution-ary optimization of the ATP-binding protein, wecharacterized 12 single point mutants of the foldingoptimized protein (Figure 1(b)). These mutationswere designed to individually revert each of these12 positions to their original residue observed in theancestral protein. We also tested two additional

Figure 2. Thrombin cleavage assay for protein solubility.separate the individual ATP binding proteins from the maltosesolubility at 2 mg/ml concentration over the course of seven dwas determined by SDS–PAGE analysis. (c) The presence or anoted with the following abbreviations: − (no precipitate), + (cases the presence of observable precipitate correlated with th

single-point mutations, R4Q and K6R. R4Q waschosen because the protein with the highest ATPaffinity generated from the folding optimizedselections differed from the consensus sequenceonly by this change.5 The K6R mutation was chosenfor analysis because of the unusual change from K6in the ancestral protein to R6 in the bindingoptimized protein, then back to K6 in the foldingoptimized protein. Comparison of the dissociationconstants for each mutant protein with the foldingoptimized consensus sequence (Figure 1(b)) en-abled us to determine which amino acid substitu-tions had the greatest effect on ATP binding affinity.Of the 12 mutations, the revertants R19W and K21Eresulted in a ten- and fivefold decrease in ATPaffinity, respectively, relative to the folding opti-mized consensus sequence. Five other reversionmutations (V10I, N29T, S38C, S41Y and V50D) andK6R showed much smaller drops (twofold or less)in ATP binding affinity. Although the significanceof these latter mutations is difficult to assessindividually, if all of the effects are real andindependent, their combined influence could be asmuch as ninefold.

Effects of mutations of protein solubility

We wished to understand the contribution of eachof the mutations that arose during the evolution ofthe optimized ATP-binding protein to the solubilityof that protein. The solubilities of the ATP bindingproteins were tested by proteolytically separatingthe fusion constructs into MBP and the free ATPbinding protein (Figure 2(a)), incubating with orwithout ATP for up to seven days, pelletingaggregated protein and examining the remaining

(a) MBP fusion proteins were cleaved with thrombin tobinding protein. (b) MBP fusion proteins were assayed forays. The presence or absence of free ATP binding proteinbsence of insoluble precipitate in the reaction mixture wassome precipitate), and ++ (significant precipitate). In moste absence of soluble protein in the gel.

Table 1. Thermal stability of the folding optimizedprotien

Entry Sequence Length T50 (°C)

1 18–19 81 562 FOB 81 763 FOB 64 744 FOB (K21E) 64 735 FOB (N29T) 64 786 FOB (N36S) 64 587 FOB (H47Y) 64 63


soluble ATP-binding protein by analyzing bandintensities on SDS–gels (Figure 2(a)). Comparison ofthe ancestral, binding optimized, and foldingoptimized proteins (Figure 2(b)) revealed that onlythe folding optimized protein remained soluble afterseven days at room temperature, while the ancestraland binding optimized proteins formed visibleprecipitates during the first 24 h and becomecompletely insoluble after three days. Surprisingly,the core domain of the divergent protein 18–19 alsoformed a visible precipitate after 24 h and wasalmost completely insoluble by three days. How-ever, in the presence of 10 mM ATP (SupplementaryData), protein 18–19 (but not the ancestral andbinding optimized proteins) remained soluble forgreater than seven days. Only the folding optimizedprotein remained stably folded and soluble in theabsence of ATP. Thus, differences in the amino acidsequence between the folding optimized protein andits ancestral forms, as well as the 18–19 variant, mustcontribute to the observed stability of the protein.Analysis of the single amino acid revertant

proteins (Figure 2(b)) indicated that five (R19W,S38C, K40N, S41Y, and V50D) formed visibleprecipitates within three days in the absence ofATP. The least soluble single reversion mutantprotein was S41Y. This is the only amino acidsubstitution to result from two genetic mutations inthe DNA sequence (UAC to AGC). Thus, optimiza-tion of the ancestral protein required a rare doublemutation to change this amino acid, supporting theimportance of a Ser residue at this position. Thechange from the ancestral W19 to R19 was notedabove as the mutation with the strongest effect onATP binding. Two of the remaining changes in theancestral sequence (Y41, D50) to the optimizedsequence (S41, V50) also affect ATP binding, but toa lesser extent. It is also interesting to note thatalthough the K6R version of the folding optimizedprotein was largely soluble after seven days, a slightprecipitate was observed (Figure 2(c)). This modestchange in solubility may be why the foldingoptimized protein reverted to K6 rather than retainthe R6 seen in the binding optimized protein and inthe 18–19 variant.

Effects of mutations on thermal stability

In addition to ligand binding affinity and proteinsolubility, we also investigated the effects of evolu-tionary optimization on the thermal stability of thefolding optimized protein by monitoring changes incircular dichroism (210 nm) as a function oftemperature. Although each of the proteins exam-ined gave sigmoidal curves consistent with a singletransition between the native and denatured states,none of the melts were reversible. Nevertheless, wewere able to determine the relative thermal stability(Table 1) of each protein by comparing the meltingtransition (T50) for protein 18–19, FOB, and foursingle-point reversions found to remain soluble afterremoval of theMBP protein affinity tag.Wewere notable to obtain thermal stability data for the F3Y,

V10I, and S63N reversions, which remained solublein the time dependent precipitation assay, due tolimited expression and decreased solubility atelevated temperatures. Since protein 18–19 was notsoluble as a 64 amino acid construct, we measuredthe thermal stability of a longer 81 amino acidconstruct that was similar to the previously crystal-lized sequence. Protein 18–19 was found to be muchless thermally stable, with a T50 20 °C lower than thefolding optimized protein. The additional amino-terminal residues that keep protein 18–19 solubleapparently do not contribute greatly to the thermalstability of the folding optimized protein, since both64 and 81 amino acid constructs yielded nearlyidentical T50 values (76 °C versus 74 °C, respec-tively). Of the four individual revertant mutationsexamined, N36S has the greatest effect on thermalstability, as this reversion lowered the T50 from 74 °Cto 58 °C. The H47Y reversion also significantlyreduced the T50 to 63 °C. No appreciable change wasobserved for the K21E and N29T reversions(T50=73 °C and 78 °C, respectively).

Structure and dynamics of the folding optimizedATP-binding protein

The crystal structure of protein 18–19 provided thefirst look at the structure of the ATP bindingprotein.3 However, the folding optimized proteindiffers from protein 18–19 at 12 out of 62 positions inthe core binding domain. In order to ensure that theobserved biochemical data were not the result ofgross structural changes, we used NMR spectro-scopic data to calculate the tertiary structure of thefolding optimized protein. Resonances were as-signed for 58 of the residues of the folding optimizedprotein (Figure 3). The unassigned amino acidresonances were Gly1, Phe3, Pro7, Pro15, Val20,and Asn22. In addition, the assignments for residuesSer2, Arg4, Val5, and Lys6 are tentative, since fewconnectivities were observed. Excluding the prolineresidues, the unassigned residues correspond toloops and turns connecting regions of secondarystructure. The inability to identify resonances withinthese regions and the lack of connectivities viathrough bondmagnetization transfer for the remain-ing resonances of the amino terminus are consistentwith conformational flexibility, which is typical ofthe termini and loop regions of proteins. Thechemical shifts and calculated dihedral angles ofthe assigned backbone resonances were consistent

Figure 3. 1H-15N HSQC of the folding optimized protein at 600 MHz. Resonances are labeled with theircorresponding residue number.


with the secondary structure seen in the crystalstructure of protein 18–19 with the exception of theloss of an amino-terminal α-helix and the presenceof an additional β-strand at the carboxy terminus.The N-terminal half of the amino-terminal α-helixpresent in the crystal structure of protein 18–19derived from a FLAG-tag used for protein purifica-tion, whereas our investigation of the foldingoptimized protein used only the core domain ofthe protein. We were able to avoid extra sequencesfrom protein tags because of the increased solubilityof the core domain of the folding optimized protein,which allowed the structure and function of theprotein to be evaluated without the influences ofadditional amino acid residues. The secondarystructural elements were further confirmed byNOE assignments with the observance of character-istic intra-helical dNN(i, i+2), dαN(i, i+2), and dαN(i, i+3)NOEs and long-range inter-strand NOEs for theβ-sheet. Taken together, the secondary structure ofthe folding optimized protein consisted of oneα-helix (K32-G42) and a four-stranded antiparallelβ-sheet composed of β1 (W18-K21), β2 (H24-Y28),β3 (H49-L53) and β4 (D57-F61).The RDC data also confirmed the similarity in

tertiary structure between the folding optimizedprotein and protein 18-19 between residues 8 and 53with a correlation coefficient of ∼0.97. Long-rangeconstraints were derived from 3D 13C- and 15N-NOE spectroscopy (NOESY) heteronuclear singlequantum coherence (HSQC) spectra and used tocalculate the structure of the folding optimizedprotein. The final structure had no NOE violationsgreater than 0.5 Å and no dihedral angle violationsgreater than 5°. The structure was evaluated withMolProbity6 and found to have 98% of its residueswithin the Ramachandran favored region for the

portions of the folding optimized protein that werestructured. The value was 85% when consideringthe entire molecule. The backbone pairwise rmsdvalue for the family of structures over regions ofsecondary structure was 0.6 Å and from residues 8 to61 it was 1.3 Å. The backbone rmsd between thefolding optimized protein and protein 18–19 betweenresidues 8 and 53 was 1.7 Å (Figure 4).Despite being an abiotic protein, the folding

optimized protein shows many characteristics of anatural protein fold, including regular secondarystructure and a compact globular tertiary fold(hydrodynamic radius=1.8 nm). The average T1,T2, and heteronuclear NOE values of 0.51 s, 0.11 s,and 0.86, respectively, are consistent with a rigid,monomeric, and globular fold for a protein of7.5 kDa and further confirmed the rigidity of thecarboxy-terminal region of the folding optimizedprotein (Figure 5).

Discussion

The in vitro evolutionary optimization of a lowaffinity ancestral ATP binding protein into a wellfolded, high affinity protein occurred through theaccumulation of 12 amino acid substitutions. Wehave investigated the affect of each of thesesubstitutions on ATP affinity and folding stabilityby examining single-point revertants at each site inthe core-binding domain of the native protein. Twoof the mutations (F3Y and S63N) have no measur-able effect on either ATP affinity or protein stability,and as far as we can tell are completely neutral. Ofthe remaining ten mutations, two affect ATPbinding strongly and five have possible weakeraffects. In addition, seven mutations affect folding,

Figure 4. NMR solution structure of the folding optimized protein. (a) Family of structures of the folding optimizedprotein. (b) Ribbon diagram of the NMR structure of the folding optimized protein (blue) superimposed against thepreviously determined X-ray crystal structure of protein 18–19 (red).


as evidenced by solubility and thermal stabilityassays, and four of these affect both binding andfolding. We will discuss these mutations in thecontext of the structure of the folding optimizedprotein.Despite being a synthetic, abiotic protein, this de

novo evolved protein contains all of the hallmarks ofa naturally occurring globular structure. The foldingoptimized protein adopts an α/β-fold characterizedby a four-stranded antiparallel β-sheet and one α-helix. The burying of a hydrophobic surface areaupon binding of the adenine nucleobase and thetetrahedral coordination of four invariant Cysresidues presumably drives the tertiary structure toa folded state. The first three strands of the β-sheetare composed of residues with high β-sheet propen-sity (Val, Ile, Tyr, and Trp)7 and have a standardarrangement of alternating hydrophobic (W18, V20,L25, I27, V50, and W52) and hydrophilic (R19, K21,H24, R26, Y28, H49, and D51) residues.8 Severalresidues within the hydrophilic face of the β-sheet(R26, Y28, H49, and K21) mediate polar interactionswith the ATP ligand. The CXXCXnCXXC zinc-binding site, although a common motif in manyproteins,9 and remarkably similar to the ‘‘treble-clef’’zinc-binding structure, is unique in the sense that noother nucleotide binding protein domains are zincnucleated structures.4 Comparison of the three-dimensional structure of the folding optimizedATP-binding protein with other nucleotide bindingproteins, such as those containing the well-charac-terized Rossmann fold, reveals that while the ATP-

binding protein adopts a completely different pro-tein fold, the process of in vitro evolution hasultimately led to the rediscovery of some of thesame ligand binding contacts as those previouslydiscovered by nature.10 For instance, the adeninenucleobase (Figure 6) is precisely positionedbetween two aromatic side-chains in a readilyobservable pi-stacking interaction and the α and β-phosphate groups of ATP coordinate one or morepolar side-chains. Such interactions appear in mostknown nucleotide binding proteins.11 One reason forthis might be that there are only a limited number ofways in which certain types of chemical function(such as ligand-binding affinity) can be achieved, buta large number of ways in which proteins can fold toachieve this function.The most dramatic difference between the solu-

tion structure of the folding optimized protein andthe crystal structure of protein 18–19 is the presenceof a fourth β-strand in the folding optimizedprotein. However, whether this represents a truestructural difference between these two proteins ispresently unclear. Support for altered structuresstems from the fact that there are sequence diffe-rences between these two proteins in this short five-residue β-strand. For example, the construct usedfor our solution structural analysis had Ala and Pheat positions 58 and 61, respectively, whereas thecrystallographically characterized protein con-tained Ser58 and Ile61. Furthermore, one of theevolved positions within β3, V50 for the foldingoptimized protein and D50 for ancestral, binding

Figure 5. T1, T2, and heteronuclear NOEs obtained forthe folding optimized protein.


optimized, and 18–19 proteins, may contribute tothe stabilization of the β-sheet (see below), andthus the stabilization of β4. Nevertheless, the an-cestral and binding optimized consensus sequencesare identical to that of the folding optimizedprotein within β4, and so loss of rigidity of thecarboxy-terminus may be a peculiarity of protein18–19. Another possibility is that the crystal state ofthe protein does not reflect its folding properties insolution, as has been occasionally observed.12

Further mutagenic and spectroscopic studies arenecessary to definitively characterize the structuraldifferences between the different ATP-bindingproteins.One of the most remarkable features of the

evolutionary optimization of the ATP-binding pro-tein is that the basic protein structure, and the coreprotein–ATP interactions, were all present in the

ancestral sequence, i.e. the original sequence presentin the random library. All of the mutations that affectATP affinity affect peripheral aspects of ATP bind-ing. The two revertant mutations (i.e. changes fromthe optimized to the ancestral state) with thestrongest deleterious effects on ATP binding, R19Wand K21E, have clear structural interpretations interms of interactions with the triphosphate of theATP ligand. The change from the ancestral W19 toR19 was first observed in the binding optimizedprotein sequence, and replaces a hydrophobicresidue with a positively charged side-chain thatinteracts with the phosphates of the nucleotide. Thisposition is K19 in protein 18–19, and the ε-aminogroup of this lysine H-bonds with the α and β-phosphate groups of ATP in the crystal structure.3

The original E21 of the ancestral and bindingoptimized proteins evolved to K21 in the foldingoptimized protein and protein 18–19. K21 appears tohydrogen bond with the γ phosphate of ATP, sincestructural calculations placed the εN of Lys21 within3.4 Å of the γ phosphate of ATP (Figure 6). Moregenerally, the region of the protein surface definedby positions 19 and 21 has evolved an electroposi-tive patch that is complementary to the highnegative charge of the triphosphate of bound ATP(Figure 7(a)). Mutations at five additional sites haveweaker effects on ATP binding (V10I, N29T, S38C,S41Y and V50D). The residues at these sites do notinteract directly with the ATP ligand, either in thecrystal structure or in our solution structure of thefolding optimized protein. These mutations maylead to indirect effects on the positioning ofneighboring residues, or may be due to alteredfolding stability in the case of S38C, S41Yand V50D.It is interesting to note that if all of these mutationsact independently on ATP binding, the combinedeffect on the Kd would be roughly 500-fold,consistent with the observed change in Kd from theancestral protein (N250 μM) to the folding optimizedprotein (500 nM).The five single-site revertants (R19W, S38C, K40N,

S41Y, V50D, from the optimized to the ancestralstate) that decreased protein solubility are readilyexplainable from the structure of the foldingoptimized protein. Three of these changes (R19W,K40N, and S41Y) significantly decrease the hydro-philicity (Figure 7(b)) of the protein surface. Position19, however, is unique in that it contributes both toimproved protein solubility and ATP affinity. In theabsence of ATP this residue is likely to be solventexposed, and so R19, as found in the folding andbinding optimized proteins, is more favorable thanthe W19 in the ancestral sequence. Following ATPbinding, R19 hydrogen bonds to the α and β-phosphate groups of ATP, as discussed above. TheK40N results in loss of a surface charge, and S41Yincreases the exposed hydrophobic surface area.S38C replaces a cysteine thiol on the protein surface,where it could potentially lead to dimerization andsubsequent unfolding and further aggregation. Theinfluence of position 50 (V50D) is more complex. Inthe crystal structure of protein 18–19, there is a salt

Figure 6. The adenine-bindingpocket present in theNMRstructureof the folding optimized protein.Residues that define the ATP-bind-ing site are labeled. The side-chainamino-groupofK21 iswithinhydro-gen-bonding distance of the γ-phos-phate of ATP, an interaction notpresent in the ancestral and bindingoptimized proteins.


bridge between residues D50 and R6. However,because R6 is located in the amino-terminal α-helix,this interaction is lost in the folding optimizedprotein. Since position 50 is on the hydrophobic faceof the β-sheet it may be more energetically favorableto have a hydrophobic residue at this position,rather than a charged polar residue. Indeed, for thefolding optimized protein, the proximity of Val50 inβ3 and Ile27 of β2 is seen via NOEs between the γand δ protons of the Ile side-chain and the backboneamide of Val50. This would account for the presenceof V50 in all of the clones examined in the output ofthe folding optimized selection. However, whetherVal50 stabilization is sufficient to allow for theformation of β4 requires further experimentalevaluation. The possible influence of Val50 on β4can been observed by NOEs between this residueand Lys59 of β4. For example, NOEs between the αand γ protons of Val50 and the α and δ protons ofLys59 were detected. Interestingly, different muta-tions are found at three of these five positions inprotein 18–19 (K19, D40, I41), suggesting that thereare different ways of optimizing the functionality ofthe ancestral protein.The thermal stability data show that several

residues have important effects not detected by thebinding and solubility assays. The N36S reversion,for example, results in a significant drop in thermalstability (58 °C versus 76 °C), probably due to theloss of the Asn36 hydrogen bond to the backbonecarbonyl of K32. The interaction of N36 and K32 issupported by a number of NOE interactionsobserved between these two residues, includingNOEs involving the side-chain amide of Asn36. TheH47Y reversion, which also causes a significant dropin thermal stability (63 °C versus 76 °C), appears tode-stabilize the evolutionarily optimized protein byloss of a hydrogen bond between the N-ε2 position

of His47 and the Ser38 side-chain. One of the moreinteresting results to come from these experimentswas the observation that the K21E reversion had nomeasurable effect on protein folding stability. Thiswas somewhat surprising given the dramatic effectthat this mutation has on ATP binding affinity, anddemonstrates that ligand binding and proteinfolding are not thermodynamically coupled. Takentogether, the thermal stability data highlights theimportance of hydrogen bonding and side-chainpacking in protein folding stability.All previous examples of the use of directed

evolution to optimize protein function have startedwith biological proteins that have a long evolu-tionary history; typically the optimization is withrespect to a new environment or a new function. Incontrast, our work started with a sequence isolatedfrom a large collection of synthetic, random proteinsequences. When we began our efforts to optimizethe initial, poorly functional sequence, it was notclear a priori that the sequence could be greatlyoptimized. or example, most random sequences withmodest degrees of function might quickly reach alocal optimum that cannot be surpassed withoutmajor structural changes. The fact that our primordialATP-binding protein could be so extensively opti-mized, both with respect to affinity and with respectto protein stability, demands explanation in terms ofthe distribution of function in sequence space. Onehypothesis that is consistent with our observationsand with the general history of protein mutagenesisstudies is that peaks in the protein fitness landscapeare steep, in the sense that they consist of relativelyfew highly fit sequences, and vastly more sequencesthat are less fit. A randomly chosen sequence with alow level of functionality is therefore most likely tocome from the base of a peak in the fitness landscape,and should therefore be optimizable by directed

Figure 7. Surface view of theancestral, binding optimized, andfolding optimized ATP binding pro-teins. (a) A positive electrostaticpatch evolved during the course ofthe selections that improved ATPaffinity. Positive, negative, and neu-tral regions are shown in blue, red,and white, respectively. Structuresrepresent residues 8–54. (b) Surfacerepresentation of the folding opti-mized protein showing mutationsresulting in increased hydrophili-city (red) and increased hydropho-bicity (yellow) based on Kyte–Doolittle hydropathy data.41 Topand bottom images refer to frontand back views, respectively. Struc-tures represent residues 8–62.


evolution. If most peaks in the fitness landscape havethe same or similar steepness, then sequences fromnear the base of a very high peak (i.e. very goodoptimal function) may greatly outnumber sequencesof similar activity that are near the top of lower peaks.

If this hypothesis is correct, then it should be possibleto significantly optimize most randomly selectedfunctional sequences. Testing this hypothesis willclearly require the selection and optimization ofmanyindependent examples of novel, func-tional proteins.


Significance

We have begun to elucidate a pathway by which aprimordial protein sequence attains increasingdegrees of functionality through the systematicaccumulation of point mutations resulting from invitro evolution. Through biochemical and structuralinvestigation of the ATP binding protein we havebeen able to suggest explanations for how changesat these positions contribute to improved solubilityand ATP affinity. Enhanced ATP affinity results fromthe emergence of a patch of positive charge densityon the protein surface, while improved proteinsolubility appears to result from mutations thatincrease the hydrophilicity of the protein surfaceand thereby decrease protein aggregation. Proteinstability may be further improved by an additionalmutation that stabilizes the hydrophobic face of theβ-sheet of the protein.The solution structure of the first synthetic protein

evolved from a library of random amino acidsequences more faithfully reflects the true structureof the ATP-binding protein, at least with respect toregions that independently evolved without theinfluence of flanking sequences. The observation of apreviously undetected β-strand presents an oppor-tunity to further probe the association between theevolution of ligand binding, protein stability, andprotein solubility with the appearance of an addi-tional region of secondary structure.In vitro directed protein evolution provides a

novel approach to understanding the changesnecessary to evolve an ancestral protein from aweakly folded state to a well-folded three-dimen-sional structure. The structure of the de novo evolvedATP binding protein reveals the exploitation of thesame strategies used by natural proteins to stabilizetertiary folds and to recognize ligands. The applica-tion of directed evolution provides an excitingopportunity to build new abiotic proteins withtailor-made functionality.

Materials and Methods

Protein expression and purification

Individual ATP binding protein (ABP) sequences wereconstructed by PCR (see Supplementary Data, Figure S1,Tables S1 and S2) and inserted into the pMal expressionvector (pIADL14) between BamHI and HindIII restrictionsites. The resulting C-terminal fusion proteins have athrombin cleavage site (LVPRGS) separating the MBPfrom the ATP binding protein (ABP).13 Plasmid DNAwastransformed into One-shot Chemically Competent E. coliTOP10 cells (Invitrogen) and sequenced to verify eachmutation (ASU Core Facility). Plasmid DNA containingthe correct mutation was transformed into E. coli BL21(DE3) strain (Invitrogen). Bacteria were grown in LBmedium supplemented with 0.1 mM ZnSO4 at 37 °C to anA600 of 0.8 and induced with 1 mM IPTG. Following anadditional 4 h of growth, bacteria were harvested bycentrifugation and re-suspended in 10 ml of phosphatebuffer (25 mM NaH2PO4 (pH 8.0), 250 mM NaCl). Cells

were lysed by sonication in the presence of 1 mg/mllysozyme. The crude lysate was clarified by filtrationthrough a 0.45 μ PES filter (Millipore). MBP-fusionproteins were bound to an amylose column (New EnglandBiolabs), washed with phosphate buffer and eluted withphosphate buffer supplemented with 10 mM maltose.MBP-fusion proteins were concentrated using an Amiconultrafiltration device (Millipore). Protein concentrationwas determined by UV-spectroscopy at 280 nm usingmolar extinction coefficients calculated for each sequence.The yield of pure MBP-ABP fusion protein obtained froma 250 ml culture of induced E. coli was ∼10–15 mg.

Dissociation constants

Equilibrium dissociation constants (Kd) for purifiedMBP-ABP fusion proteins were measured by equilibriumultrafiltration.14 Apparent Kd values were measured usingtrace [γ-32P]ATP (Amersham Biosciences) and a series ofconcentrations of MBP-ABP fusion protein spanning theKd app. The data were iteratively fit (through non-linearregression) using the computer program Deltagraph 4.0(Red Rock Software) to the y=b+ c((x/(x+Kd app)), where yis the experimentally measured value (top counts-bottomcounts/top counts), x is the protein concentration, brepresents non-specific binding to the protein and/orfilter, and c is the maximum fraction of counts that can bebound.

Solubility assay

MBP-ABP fusion proteins (2 mg/ml) were incubatedovernight at room temperature with thrombin (1 mg/ml;Sigma) to cleave the MBP-ABP fusion into separateproteins. The relative amount of free ABP that remainedin solution was monitored over time (one, three, five andseven days) by centrifuging the reaction mixture andanalyzing a small aliquot (5 μl) of the supernatant on a10%–20% gradient SDS–PAGE (Tris-Glycine buffer sys-tem, BioRad). Comparing the band intensity of eachmutant protein to the folding optimized protein provideda direct assessment of relative solubility. The presence orabsence of insoluble precipitate was noted with thefollowing abbreviations: - (no precipitate), +equation(some precipitate), and ++ (significant precipitate).

Circular dichroism

Circular dichroism (CD) spectra were acquired using anAviv CD Spectrometer model 202. Spectra were recordedin phosphate buffer without exogenous ATP by monitor-ing the wavelength dependence of [θ] in 1 nm incrementswith a sampling time of 10 s. Melting curves weregenerated by monitoring the change in wavelengthdependence of [θ] at 210 nm over a temperature gradientof 24 °C –90 °C. T50 values were determined by curvefitting using the program Deltagraph 4.0 (Red RockSoftware).

15N and 13C-isotope labeled protein

Samples used for NMR were purified from cells grownin M9 minimal media supplemented with 15NH4Cl and[13C]glucose (Cambridge Isotopes). The protein constructused for NMR measurements was a truncated version ofthe folding optimized protein analogous to clone B6 (ABP

†http://www.pymol.org

Table 2. Structural restraints and statistics

NMR restraintsNumber of distance restraints

Total inter-proton 677Sequential (i, i+1) 349Medium-range from (i, i+2) to (i, i+5) 202Long-range (Ni, i+5) 126

Number of dihedral anglesTotal 60Phi 30Psi 30

Total number of contraints 737Total number of constraints per residue 11.5

Structural statisticsFavored region (%) 77Additional allowed region (%) 21Average pairwise rmsd

Backbone (Å) 1.3Heavy (Å) 1.9


B6-62), except that a carboxy-terminal non-Zn ligatingcysteine (C58) was replaced with an alanine. NMRsamples were extensively dialyzed against 10 mM sodiumphosphate, 50 mM KCl, 0.5 mM ATP (pH 6.5) andconcentrated with a centricon YM-3 (Amicon) to ca0.3 mM. Samples used for residual dipolar couplingmeasurements were partially aligned with 10 mg/ml Pf1phage (ASLA biotech).

NMR spectroscopy and structure calculations

NMR spectra were either acquired on a Bruker 600MHzequipped with a cryo-probe at the MIT Francis-BitterMagnetic Laboratory or on a Bruker 700 MHz equippedwith a cryo-probe at Bruker BioSpin AG, Fällanden,Switzerland. Spectra were either processed withNMRPipe15 or Bruker TopSpin 1.3 and analyzed withCARA16 and NMRDraw. Backbone resonances wereassigned via HNCA17 and HNCACB18 at 600 MHz andHNCO19 at 700 MHz. Side-chain assignments were madeby a (H)CCH-total correlated spectroscopy (TOCSY)20

(with a mixing time of 22 ms) and 1H -15N-TOCSY-HSQC(with a mixing time of 60 ms) at 700 MHz. HNHA21 and1H–15N-NOESY-HSQC22 spectra at 700 MHz were usedfor torsion angle calculations. The HNHA spectrum wasused to calculate backbone dihedral ϕ angles from 3JHNHαcoupling constants determined from the appropriateKarplus relationship.23,24 The ψ dihedral angles werecalculated from NOE peak intensity ratios of dαN(i-1, i)and dNα(i, i) in a 1H–15N-NOESY-HSQC spectrum.25

Dihedral angles were additionally analyzed by correla-tions with chemical shifts via TALOS.26 Residual dipolarcouplings were calculated from 2D IPAP 15N-1H HSQC27

spectra by comparing JNH couplings between isotropicand partially aligned ABP FOB samples. The RDC valueswere then compared with those expected from the crystalstructure of protein 18–19 (PDB ID, 1UW1) using PALES.28

NOEs were measured from 3D 1H–13C-HSQC-NOESY22

(with a mixing time of 120 ms) and 3D 1H–15N-HSQC-NOESY22 (with a mixing time of 120 ms) spectra collectedat 700 MHz.Spectra for dynamics analysis were carried-out at

700MHz. A series of 1H–15N HSQC spectra were collectedwith flip-back29 and phase-sensitive30–32 pulse sequences.15N longitudinal relaxation rates, T1, were measured withdelays of 5.0, 100, 200, 400, 600, 1000, and 1500 ms. 15N

transverse relaxation rates, T2, were measured with delaysof 16.5, 49.5, 99 (2×), 165, and 398 ms using a τm of 900 μs.Heteronuclear 1H–15N NOEs were measured by takingthe ratio of peak volumes acquired with and without 1Hsaturation.29 Relaxation rates T1 and T2 were calculated byfitting cross-peak intensities (I) as a function of the delaytime (t) to a single-exponential decay (I= Ioe

(t/T), where T iseither T1 or T2).Structures were calculated with a hybrid distance

geometry - simulated annealing protocol33,34 withXPLOR-NIH.35 Parameter files for ATP and zinc werefrom HIC-Up.36 Distance restraints for the ATP and Zn2+

were taken from the crystal structure of protein 18–19(PDB ID, 1UW1). A total of 677 distance restraints wereused for the folding optimized protein, including 349sequential (i, i+1), 202 medium-range (from (i, i+2) to(i, i+5)), and126 long-range (greater than (i, i+5)) constraints(Table 2). Two-hundred structures were calculated togenerate a family of 20 structures. The hydrodynamicradius of the folding optimizedproteinwas calculatedwithHYDROPRO37 and theoretical T1, T2, and heteronuclearNOE values were calculated with HYDRONMR.38 Modelsof the ancestral and binding optimized proteins werecalculated with MODELLER 8v0.39 Structures were visua-lized with PyMol† andMOLMOL.40

Protein Data Bank accession code

The coordinates of the folding optimized protein havebeen deposited in the RCSB Protein Data Bank (PDB) withaccession code 2P0X.

Acknowledgements

This work was supported by grants from theNASA Astrobiology Institute (NNA04CC12A) (toJ.W.S.) and from the BioDesign Institute at ASU (toJ.C.C.). J.W.S. is an Investigator of the HowardHughes Medical Institute. S.S.M. is a NIH post-doctoral fellow (F32 GM07450601). We thank DrCristina Del Bianco and Dr Karel Kubicek forproviding helpful scripts.

Supplementary Data

Supplementary data associated with this articlecan be found, in the online version, at doi:10.1016/j.jmb.2007.05.062

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Edited by F. Schmid

(Received 15 December 2006; received in revised form 1 May 2007; accepted 22 May 2007)Available online 26 May 2007

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