Physicochemical and structural implications for molecular recognition in immobilized metal affinity chromatography

METHODS:

A Companion

to Methods

in Enzymology

4, 14-24

(1992)

Physicochemical and Structural Implications for Molecular Recognition in Immobilized Metal Affinity Chromatography Nadir T. Mrabet Laboratoire d%nzymolo&e _ Blvd. des Aiguillettes, B.P.

et de G&e 239, 54506

Ghze’tique,_ URA-CNRS Vandczuvre-LB-Nancy,

457. Universite’ France

A precise understanding of the mechanisms that govern selectivity in protein adsorption to metal-chelate complexes is required to provide a rational framework for predicting protein behavior in immobilized metal affinity chromatography (IMAC). This article highlights the contribution of solvent-linked parameters in ligand/ligate interactions in IMAC and, hence, brings new clues to selectivity engineering. This leads to the design of a rapid and convenient protocol for the purification of recombinant Actinoplanes missouriensis o-xylose isomerase (Xl) and engineered variants produced in fscherichia co/i on copper-loaded Chelating Sepharose Fast Flow, an agarose-based matrix derivatized with iminodiacetic acid (IDA) groups. The elucidation of the molecular mechanism by which Xl recognizes and interacts with the immobilized metal-chelate complex, IDA-Cu(ll), is further made possible by a combination of X-ray structure analysis, molecular modeling, genetic engineering, and mutant characterization. Substitution of lysine for a selected surface histidine residue at position 41 yields a mutant enzyme with near-wild-type properties. The same mutation, however, is shown to completely abolish Xl adsorption onto IDA-Cu(ll), and hence directly implicates histidine 41 as the predominant protein ligand to the copper-chelate complex. This study not only provides new insights into protocol design for purifying proteins by IMAC, but also describes structural and modeling approaches to analyzing protein surface properties in relation to molecular recognition events. 0 1992 Academic press, I~C.

Protein purification is likely to constitute a bottleneck in the study of structure/function relationships 14

de Nancy

I, Faculte’

des Sciences,

in a protein engineering program in which multiple mutants need to be compared. Indeed, most purification methods consist of one or more initial concentration steps followed by a series of conventional chromatographic procedures. Such traditional multistage protocols may span several days, result in large yield loss, contribute to protein sample “aging” and denaturation, and involve high operational costs. A convenient purification scheme must be simple, fast, suitable for a large number of the protein variants being investigated, and economical. This may explain the success of affinity-based methods which take advantage of specific complex formation between a solid-phase immobilized ligand and a complementary biomolecule, so that the latter remains selectively adsorbed while contaminants are washed away (l-3). One can, however, encounter major drawbacks in separation protocols that use either biospecific affinity ligands (4) or immunoadsorbents (5). Immobilized metal affinity chromatography (1MAC)l (6, 7) may appear conceptually very rudimentary when compared to genuine biospecific-ligand i Abbreviations used: IMA, immobilized metal affinity; IMAC, IMA chromatography; IDA, iminodiacetic acid, IDA-Me(II), chelate complex between IDA and any divalent metal ion; IDA-Cu(I1) IMAC, IMAC with IDA-Cu(I1) as affinity ligand; Im, imidazole; FPLC, fast protein liquid chromatography; XI, D-xylose isomerase; Mops, 4morpholinepropanesulfonic acid; Mes, 4-morpholineethanesulfonic acid, MMA, buffer solution of defined concentration containing equimolar amounts of Mops, Mes, and acetic acid; ASA, accessible surface area, defined as the area of the surface generated by the center of a spherical probe as it rolls over the van der Waals surface of a given molecule; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis. 1046~2023/92 $5.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

MOLECULAR

RECOGNITION

affinity chromatography, and it certainly lacks its exquisite specificity and high binding affinities. Yet, IMAC offers a number of outstanding advantages for protein purification (7-10). Therefore, mechanistic insights into how protein binding selectivity for immobilized metal chelates can be modulated should further stimulate the exploitation of IMAC for the purification of proteins. The present article is concerned with the physical, chemical, and structural aspects of molecular recognition of immobilized metal-chelate complexes by proteins. A number of concepts are reviewed to provide rational guidelines for protein purification by IMAC. Use of IMAC in a predictive manner is illustrated by the single-step purification of D-xylose isomerase from crude cell lysate. The combination of protein structure analysis, molecular modeling, and site-directed mutagenesis is further shown to constitute a powerful approach to understanding molecular recognition mechanisms at the atomic level.

CHROMATOGRAPHIC PROCEDURES IN IMAC Preparation

of Solvents

The choice of solvents in IMAC will depend on the protein to be separated (11). The requirement for the availability of an electron-donor group on the protein surface that is at least partially unprotonated would dictate that the sample be applied on chelating gels at alkaline pH. Review of the literature, however, reveals successful peptide or protein adsorption at pH values as low as 5 (12,13). Moreover, 12 of 32 papers published between 1975 and 1992 mention pH values below 7; averaging yields a mean pH value of 5.9 (1 SD = 0.5). It turns out that this value is very close, if not identical, to that of the pK, of the solvated side chain of histidine, which is about 6 (14). At pH near 6, half of the surface histidine side chains are unprotonated, and hence able to establish coordination bonds to chelated metals. “Stepwise” binding to the chelating matrix is expected to further shift the equilibrium toward the formation of unprotonated species and ultimately result in complete protein adsorption. In contrast, other amino acids that contain potential electron-donor groups have much higher pK, values. Thus, the thiol group of cysteine has a pK, of about 9, while a pKa of 15 can be calculated for the side-chain nitrogen atom of tryptophan using the Hammett equation (see Ref. 15). On this basis, pH values of 7 for cysteine and 13 for tryptophan would be required to displace their respective

15

IN IMAC

equilibria toward the formation of not more than 1% unprotonated species. As a consequence, this provides strong support for the proposal that histidine is the predominant ligand in IMAC and should further suggest that there is ample room for pH adjustment to improve selectivity. If surface histidine residues are present, we can predict that a pH value of 7 is high enough to ensure protein retention on IDA-Cu(I1) columns. Since ligand binding is stronger at higher pH, we can expect, however, that protein desorption will require elution conditions that are more vigorous. According to the bond length variation rules (16), binding energy increases while the donor/acceptor bond length decreases; this in turn eventually favors the formation of additional coordination bonds with other ligands in the protein that are now brought into close contact with the metal, the end result being that the coordination center becomes completely buried and hence unaccessible to competing electron-donor solutes newly introduced in the solvent. It thus becomes clear that in order to be able to control protein elution-and also yields-binding must not be too strong; instead, it should be adjusted by appropriate pH reduction and/or by increasing the concentration of a competing ligand such as imidazole in the equilibration buffer. If these approaches fail, choosing a different metal ion such as Ni2+, or Zn2+, or Co2+ may become relevant (see, e.g., Ref. 17). The chromatographic elution of proteins in IMAC can be conveniently achieved in some instances by reducing the pH of the mobile phase. As histidine becomes protonated with decreasing pH, the binding of this group to the chelated metal weakens until the protein is ultimately released to the solvent. The proton is clearly the smallest, and hence likely the best, chemical entity one can use as a competing agent, yet its effect can be significantly enhanced with other displacers, such as ammonia or imidazole, present in the solvent (18; my unpublished observations). Meanwhile, one should bear in mind that the stability of metal chelates is maintained at least through pH 5 for IDACu( II) and IDA-Ni( II) but not for IDA-Zn(I1) or IDACo(I1) (7). This further suggests that special care must be taken in the selection and preparation of solvents for proper pH control, and the buffering capacity must be maintained over the whole pH range of the gradient to prevent sudden pH fluctuations. The preparation of buffer mixtures containing equimolar amounts of Hepes (pK, = 7.48 at 25°C and 0.1 M),2 Mes (pK, = ’ All pK,, values

are obtained

or derived

from

Ref.

14.

16

NADIR

T. MRABET

6.09 at 25°C and 0.1 M), and acetic acid (pK, = 4.76 at 25°C) was previously described by Figueroa and colleagues and shown to yield a linear pH decrease from 7 to 5 as a result of high, constant buffer capacity over the pH range 4-8 (18). More recently, similar buffer mixtures were introduced (19), in which Mops (pK, = 7.15 at 25°C and 0.1 M) is substituted for Hepes to prevent the occurrence of oxidative processes that can be produced in piperazine ring-containing Good’s buffers (e.g., Hepes; 20) in the presence of copper ions (21, 22). For the purpose of reproducibility, it is good practice to standardize the preparation of chromatographic solvents. These can be readily obtained by dilution of a concentrated stock solution and appropriate pH adjustment. The stock solution should be 10 to 50 times more concentrated and prepared in a volumetric flask containing Milli-Q-grade water from correctly weighed amounts of the acidic form of high-grade buffers. The resulting solution must then be filter-sterilized through a 0.2-/*rn filter and can be stored for sufficiently long periods of time (>l-2 months) in the refrigerator. Although less prone to microbial contamination than the phosphate-based buffers traditionally used in IMAC, the solvent mixtures described above should be supplemented with antimicrobial agents such as sodium azide (0.02% final). Also, micromolar amounts of metal ion can be added to eventually compensate for metal bleeding from the chelating gel during chromatographic runs. At such low concentrations, the presence of free Cu2+ has been shown to have no influence on IMAC of several proteins and peptides (18, 19, 23). In parallel, one should prepare a stock solution (5 10 N) of sodium hydroxide, which should be stored in a dark, tightly capped bottle. The final buffer solutions can then be prepared “on the spot” by mixing precalculated volumes of buffer stock solution and sodium hydroxide to yield the desired pH, adding salt if required, bringing the mixture to the appropriate volume, and finally filter-sterilizing the final solution. A significant advantage to preparing buffers by mixing predetermined amounts of acid and base, as derived from the Henderson-Hasselbalch equation, is reproducibility in terms of buffer composition, including pH and final ionic strength, but also the convenience of bypassing the use-and often elaborate calibration-of a pHmeter. A buffer mixture made up of equimolar amounts (20 mM) of Mops, Mes, and acetic acid, and further containing 2 yM CuS04 and 0.02% NaN3 (20 mM MMA), can cover the pH range from 4 to 8. Good and reproducible pH gradients over the range 5-7 are

thus obtained by mixing predetermined amounts of 20 mM MMA, pH 7.0, and 20 mM MMA, pH 5.0. An elution protocol in IMAC may also involve using ligand exchange with imidazole, in which case it is recommended, for the purpose of reproducibility, to first equilibrate the metal-chelate resin with an imidazolecontaining buffer before applying the protein sample and developing the column (7). This recommendation is based on the fact that imidazole can form stable complexes with the chelated metal of the form chelateMe(II)(Im),. Although this point on column equilibration is correct and appropriate, it must be stressed that on chemical grounds the number of imidazole molecules participating in complex formation can vary from 0 to 3 in the case of IDA-Me(II), assuming an octahedral metal-coordination geometry. Furthermore, this number can take noninteger values depending on the concentration of imidazole, since complex formation conforms to an equilibrium reaction. Therefore, even though imidazole must be present, its initial concentration must be low enough not to compete with coordination bond formation between IDA-Me(I1) and the protein of interest. The same reasoning applies to other potential ligands also present in the equilibration solvent. Only those metal-coordination orbitals not stably captured by such ligands can become available for bond formation with the protein. One should also keep in mind that the effective concentration of imidazole (pK, = 6.95 at 25°C) is that of the unprotonated form. Consequently, unless otherwise required for specific applications, the buffer containing imidazole should have a neutral to slightly alkaline pH. Since the pH of the equilibration buffer is instead likely to be neutral to slightly acidic, this requirement should further promote the use of high-buffer-capacity mixtures of the kind described above. Column Packing As in any other chromatographic protocol, one wishes to be able to use low-pressure yet high-resolution resins. I would highly recommend the IDA-derivatized agarose-based matrix Chelating Sepharose Fast Flow from Pharmacia. This beaded material is obtained by cross-linking Sepharose with dibromopropanol, which results in highly improved rigidity, thereby allowing very high flow rates while maintaining low pressure. Moreover, high-performance separation has been achieved in the few reports that mention its use (19,24-28). Furthermore, the Chelating Sepharose Fast Flow resin is very easy to handle. Ideally, the resin should be packed in a glass column with an adjustable top plunger, e.g., of the Pharmacia

MOLECULAR

RECOGNITION

FPLC type. After being washed with water, the desired amount of gel must be resuspended in a solution of high ionic strength (aO.2 M NaCl) before packing. This follows from the fact that the IDA-gel is negatively charged (11) and electrostatic quenching is required for adequate packing. After the gel settles, the column plunger is then gently adjusted and the column is connected to the chromatographic equipment. Packing is continued at a higher flow rate (e.g., 2 ml/min) than the rate used for purification (e.g., 1 ml/min) with at least 5 column volumes of the high-ionic-strength solution and further adjusting of the plunger. The column should be washed again with the same volume of water, and finally brought to pH 5 with an appropriate buffer. Acidification of the IDA-gel is intended to prevent the formation of weak bonds with metal ions and to provide for a more homogeneous distribution of the metal-ligation states (see, e.g., Ref. 29). In addition, divalent metal ions are known to undergo hydrolysis, leading to precipitation of insoluble metal hydroxide, and use of lower pH is therefore recommended.3 The column is then saturated with a 0.2-pm filtered stock solution of the desired metal ion (e.g., 10 mg/ml CuS04 in HzO, for which the pH is -4). Excess and loosely bound metal is then removed by flushing with (i) 5 column volumes of the pH 5 buffer, (ii) 5 column volumes of the imidazole-containing buffer, (iii) 5 more column volumes of the pH 5 buffer to eliminate imidazole, and finally (iv) 5-6 column volumes of the desired equilibration buffer. After a chromatographic run that incorporates imidazole as an affinity eluent, it is important to bring the concentration of the displacer back to “equilibration” values. This requires flushing the column with at least 5-6 volumes of the equilibration buffer. A good indication that equilibration has not been achieved is provided by the premature elution of a reference protein.

IMAC BY THE RULE: ONE-STEP PURIFICATION OF RECOMBINANT Actinoplanes missouriensis D-XYLOSE ISOMERASE The Protein Microbial D-xylose isomerases (XI; EC 5.3.1.5) are large thermostable metalloenzymes that catalyze the 3 The pK values for hydrolysis of Cu’+, Co’+, Zn’+, and 8.0,8.9,9.0, and 9.9, respectively, but the onset of precipitation occurs about 2-3 pH units below the pK value (30).

Ni2+ are usually

IN IMAC

17

aldose-ketose isomerization of D-xylose into D-xylulose. Because they are able to also convert D-glUCOS2 into the sweeter D-fructose, these enzymes are used in industry for the production of high-fructose corn syrups. The XI gene from Actinoplanes missouriensis has been cloned and overexpressed in Escherichia coli as a soluble and active recombinant protein (19,31). X-ray studies of xylose isomerase from different sources show that the protein is a tetramer of identical subunits. Each subunit consists of a main domain which folds into a typical (p/c~)s triose phosphate isomerase barrel motif and a C-terminal loop involved in intersubunit contacts. The crystal structure of the enzyme from A. missouriensis was initially solved at 2.8 A resolution (32) and has since been further refined to 2.2 A (33, 34). This protein is highly negatively charged since it migrates on isoelectrofocusing gels near pH 3 (N. T. Mrabet, unpublished data). Computation of the accessible surface area (ASA) of A. missouriensis XI using the analytical algorithm SURVOL (35) integrated into the modeling package BRUGEL (36) indeed confirms that the protein with two metal-ion cofactors bound per subunit has a net solvent-exposed charge of -36 at neutral pH (19). Analysis of the X-ray structure of XI also reveals the presence of surface histidines, making this enzyme a likely candidate for purification by IMAC. The Purification Strategy IMAC is a group-specific purification method. If a purification strategy is to start from a crude protein sample such as a total cell lysate, a whole range of binding affinities for the metal-chelate complex is likely to be displayed by the various components present in the mixture. Therefore, every attempt should be made to desorb the protein of interest in the middle of an elution gradient, that is, after components with lower affinity have been eliminated, while retaining those with higher affinity. As mentioned earlier, a number of factors can come into play at this stage to fine-tune binding selectivity, some of which are further discussed below. A copper-loaded Chelating Sepharose Fast Flow column was used. The protein sample, a cell-free extract from E. coli cells expressing the recombinant D-xylose isomerase enzyme, was loaded onto the column equilibrated in 20 mM MMA, pH 6.0, 0.5 M NaCl, 0.25 mM imidazole, and further run isocratically for 20 min at a flow rate of 1.1 ml/min (Fig. 1). Several absorbance peaks are shown to elute unretained from the column (Fig. 1B). By means of a ternary gradient, both pH and NaCl concentration were reduced, before a shallow im-

18

NADIR T. MRABET

idazole gradient from 0.25 to 1.25 mM was applied (Fig. 1A). This led to the elution of a single, fully resolved, and symmetrical peak at 69 min (Fig. 1B). Desorption of cell lysate components bound with the highest af-

finities was subsequently accomplished by raising the imidazole concentration to 25 mM and the pH to 7. Purity was analyzed by 10% discontinuous SDS-PAGE (37) and staining with Coomassie blue R-250 as shown

A

0.5 7.0 0.4

25 20

0.3 -8

6.0

15 0.2

10 0.1 5 0.0 I

I

I

0

20

I

I

40

60

Retention

I

I

80

100

I

I

120

140

0

Time (min)

CL

A

B

C

D

E

CL

Hk---iHHW-HH

0

20

40

60

80

100

120

140

Retention Time (min) FIG. 1. One-step purification by IDA-Cu(I1) IMAC of recombinant A. missouriensis D-xylose isomerase enzyme produced in E. coli. The sample is an aliquot (-10 mg) of soluble supernatant obtained after streptomycin sulfate treatment of total cell lysate (see Ref. 19). (A) The measured pH and NaCl gradients. The imidazole gradient is theoretical and is based on the gradient program described below, but it was corrected for an -7-min gradient lag time (from the solvent gradient-mixing pump to the detector flow cell): (1) 49% A/50% B/l% C, isocratic for 20 min; (2) linear gradient to 35% A/64% B/l% C in 30 min; (3) linear gradient to 99% B/l% C in 15 min; (4) linear gradient to 95% B/5% C in 15 min; (5) linear gradient to 100% C in 15 min; (6) isocratic for 3 min; (7) linear gradient to 49% A/50% B/ 1% C in 10 min; (8) isocratic for 40-50 min before a new sample is injected. Flow rate is 1.1 ml/min. Solvents: A = 20 mM MMA, pH 7.0, 1.0 M NaCl; B = 20 mM MMA, pH 5.0; C = 20 mM MMA, 25 mM imidazole, pH 7.0. (B) The resulting chromatographic profile. The Chelating Sepharose Fast Flow (-8 ml) charged with CL?+ was packed in a Pharmacia HR lO/lO column connected to a Varian 5060 ternary-gradient HPLC system, equipped with a Polychrom 9060 diode-array detector and a SpectraPhysics 4290 integrator. Samples were injected by means of a l-ml loop mounted onto a Rheodyne automatic injector. (C) Discontinuous SDS-PAGE (lo%), with Coomassie brilliant blue R-250 staining, analyzing the peak fractions-labeled as in B-of the chromatogram. Fractions were collected using a Pharmacia FRAC-200 fraction collector set in the peak collection mode with a 3% cutoff; maximum fraction size was 2.2 ml. XI designates control xylose isomerase protein purified according to a traditional protocol previously described (19; see text); CL is total cell lysate before IDACu(I1) IMAC. (Adapted with permission from Ref. 19. Copyright 1992 American Chemical Society.)

MOLECULAR

RECOGNITION

in Fig. 1C. The peak at 69 min is shown to yield a single band which comigrates with the xylose isomerase standard. This peak was collected into three separate fractions (labeled C on the gel, Fig. 1C). The most prominent fraction in the center corresponds to the top of the peak and represents a total protein load of 125 pg. Assuming a sensitivity limit for the protein stain of 0.2-0.5 pg (38), the absence of any detectable protein band other than XI indicates that enzyme purity exceeds 99%. Therefore, purification of XI to homogeneity has been accomplished in a single chromatographic step. Rationalizing the Outcome of the IMAC Experiment As reviewed earlier, and in accord with the presence of surface histidines, XI adsorbs to IDA-Cu(I1) at pH 6. Binding, however, requires the presence of salt (0.5 M NaCl), a behavior that is very likely explained by the need to quench repulsive electrostatic interactions between the highly negatively charged enzyme and an affinity sorbent that is itself predominantly negatively charged (11). Interestingly, raising the imidazole concentration to 1 mM in the equilibration buffer abolishes XI retention on the chelating gel. In fact, the selection of solvent parameters (e.g., 0.25 instead of the traditionally used 1 mM imidazole) for column equilibration was guided by a number of such observations. For example, increasing the initial pH to 7 results in the retardation of low-affinity contaminants with tailing overlap onto the XI peak (19). Elution of the bound enzyme could be achieved by simply raising the imidazole concentration to 25 mM at pH 7, while maintaining the NaCl concentration at 0.5 M, by means of a traditional binary gradient. Under these conditions, however, XI is eluted late, near the end of the gradient, and is contaminated with other late proteins. This finding and the knowledge that both the enzyme and the IDA-Cu(I1) gel are negatively charged thus led to the elution strategy illustrated in Fig. lA, where XI elution is accomplished by simultaneously reducing the pH to 5 and the NaCl concentration to 0 and then slightly increasing the imidazole concentration from 0.25 to -0.4 mM. Therefore, enhancing the electrostatic repulsion between the enzyme and the chelating gel leads to the desorption of XI in a rather selective fashion, since the binding of other high-affinity components is not affected. Finally, these high-affinity contaminants could be discharged by means of a linear gradient to 25 mM imidazole at pH 7, and a new chromatographic run could be started immediately after column equilibration, without the need for the lengthy column regen-

19

IN IMAC

eration procedure involving EDTA flushing, gel washing, and metal ion reloading so often advocated by several investigators (see, e.g., Refs. 24, 28, 39). In conclusion, these results demonstrate the feasibility of rational optimization of IMAC. Recovering Enzymatic

Activity

after IMAC

Inactivation of enzymes via metal poisoning has been a perpetual burden to protein biochemists. Whether this has been, at least on occasion, an impediment to even attempting to use IMAC remains to be determined. Like other xylose isomerases, the enzyme from A. missouriensis requires metal cofactors for catalytic activity (34), the order of preference being Mg2+ > Co2+ > Mn2+. In contrast, other divalent metal ions such as Cu2+ Ca2+ Zn2+ , Ni2+, and Fe2+ are strong inhibitors (N. T. Mrabet, unpublished experiments). As a consequence, XI recovered after IDA-Cu(I1) IMAC is totally devoid of enzymatic activity. Since inhibition is observed whether or not buffers are supplemented with free copper ions, this finding implies metal ion bleeding and/or scavenging. Reactivation of XI therefore implied the elimination of Cu’+, but the metal ion appeared to be tightly bound to the enzyme. Indeed, following extensive dialysis against EDTA at pH 6, enzyme reactivation ranged from 70 to 90%, suggesting the incomplete removal of copper ions from the protein. Demetallation could, however, be markedly improved by also including NaCl to 0.5 M (N. T. Mrabet, unpublished experiments). On this basis, a protocol that called for a polishing step of chromatography on Mono-Q was devised (19). The column was equilibrated at pH 6.0 and 0.1 M NaCl in 20 mM Mes, 10 mM EDTA, and 0.02% NaN3. Under these conditions the enzyme remained bound while the column was flushed isocratically with 20 bed volumes of the starting buffer. Elution was then achieved by raising the salt concentration to about 0.4 M in 20 mM Mes, 10 mM EDTA, 0.02% NaN3, pH 6.0, to yield a fully active enzyme (19). It is therefore likely that a similar methodology that involves enzyme immobilization on an ion-exchange resin and flushing with EDTA or another chelator at sufficiently low pH succeeds as well with other proteins. The reason that the presence of salt appears to facilitate metal removal from the XI enzyme is not clear, except for the screening of possible ionic interactions between the positively charged metal ions and the highly negatively charged metal-binding sites which may not be readily accessible to EDTA.

20

NADIR T. MRABET

PROBING THE MECHANISM OF MOLECULAR RECOGNITION IN IMAC BY PROTEIN ENGINEERING A full understanding of macromolecular interactions calls at least for knowledge of the three-dimensional structures of the molecules involved at a level close to atomic resolution. The highly refined crystal structure of A. missouriensis XI (33, 34) was examined in an effort to evaluate its metal-chelate binding properties from a structural perspective, and hence pinpoint potential electron-donor groups present on its external surface. From a functional point of view, protein external surfaces play a prominent role. They are the focus of interactions with the “outside world” which includes a variety of molecules both small and large, such as solvents, substrates or inhibitors, and other proteins. As a result, these interactions require that the protein surface recognize other surfaces that have a variety of shapes, sizes, and physicochemical properties. Xylose isomerase from A. missouriensis contains 10 histidines and 5 tryptophans per subunit and is totally devoid of cysteine. Examination of the crystal structure suggests that only histidines can engage in interactions with chromatographic surfaces. Although 2 tryptophans at positions 16 and 20 are close to the “external” surface of the protein, they occur in a channel at subunit interfaces and are furthermore sandwiched between several residues (not shown). The participation of Trp-16 and Trp-20 in IMAC would therefore require significant structural reorganization to provide for increased accessibility. Although the possibility of such changes in conformation cannot a priori be excluded, the distinguished stability properties of the enzyme (33) argue strongly against their occurrence. Furthermore, the adsorption of XI onto IDA-Cu(I1) is accomplished at pH 6, providing additional grounds for excluding the participation of tryptophans. As a result, only histidines are considered further in the following analysis. Computation of surface areas accessible to solvent was performed using a spherical probe with a radius of 1.4 A, which mimics the water molecule (40), whereas accessibility to the IDA-Cu(I1) ligand attached to Chelating Sepharose Fast Flow was calculated using a larger probe sphere with a radius of 1.93 A (19). Importantly, this increase in size, which is derived from a model-built three-dimensional structure of the metalchelate complex, represents the smallest radius increment that can be reasonably derived from structure analysis (19). Therefore, loss of accessibility of a given

residue as a result of increasing radius size from 1.4 to 1.93 A is to be interpreted as a definitive result, at least on static grounds. In contrast, if accessibility to IDACu(I1) is in theory still measurable, the actual implication of the residue(s) concerned is yet to be ascertained. Table 1 lists the accessible surface areas of histidines on the external surface of the XI tetramer. The 5% cutoff threshold on the ASA for distinguishing buried from exposed residues is calculated as 9.7 A2 for histidine (41). On this basis, four histidine residues at positions 41,54,96, and 250 are found on the external protein surface and are accessible to solvent molecules with radii ~1.4 A. Increasing the probe size to 1.93 A, however, suggests that only a single residue, namely, His-41, has remained available for interacting with IDA-Cu(I1). In particular, the ASA change for His-41 in these circumstances is only -6%, which is strong evidence that the residue is protruding toward the solvent (42). In contrast, for other histidines the ASA on the external surface decreases from 34 (His-389) to TABLE

Variation

with of Histidine

Probe Size Residues

A. missouriensis

1

of the Accessible Surface on the External Surface D-Xylose Isomerase” Probe

Residue His-41 His-54 His-96 His-220 His-238 His-243 His-250 His-262 His-290 His-389

r = 1.4 A 68.67 10.47 10.80 5.48 6.90 0.06 40.99 0.57 0.01 7.94

2 + + + 4 2~ + * * +

4.06 1.21 0.92 0.26 0.27 0.04 0.69 0.11 0.01 1.05

Areas of

size r = 1.93 A 64.52 2 4.15 1 4.34 + 2.01 f 2.57 zk Absent Absent Absent Absent 5.22 f

4.13 0.48 0.75 0.18 0.33

0.68

’ Accessible surface areas (ASA, As) were calculated with the analytical procedure SURVOL (35) in BRUGEL (36) on a Silicon Graphics INDIGO graphics station, using the 2.2-A resolution crystal structure of the cobalt/xylitol-bound A. missouriensis D-XylOSe isomerase (33, 34; Brookhaven Protein Data Bank (44) entry name is IXIM), after elimination of ligands including crystallographic water molecules. Accessibility to solvent was determined using a spherical probe with a 1.4-A radius. Accessibility to the metal-chelate complex present in Chelating Sepharose Fast Flow, N-[3-((3’.methoxy-2’.hydroxypropyl)oxy)-2-hydroxypropyll-IDA-Cu(II), was instead computed using a probe sphere with a radius of 1.93 A, calculated from the model-built three-dimensional structure of this molecule as previously described (19). The ASA values listed represent averages over the four subunits (& 1 SD) and are strictly those on the external surface of the XI enzyme, hence excluding cavity surfaces. “Absent” means that the concerned residue no longer belongs to the external surface.

MOLECULAR

RECOGNITION

over 60% (histidines 54, 220, and 238). Finally, we see that four residues, at positions 243, 250, 262, and 290, are no longer components of the external surface. The most striking feature observed as a result of the increase in probe size concerns histidine 250, the second most solvent-accessible histidine in XI with an ASA of 40 A”. His-250 is indeed found to “transfer” from the external surface into an “enclosed” cavity, and hence can no longer engage in interactions with IDACu(I1) (19). This analysis is therefore a plain demonstration of the influence of probe size on the evaluation of residue accessibility. A corollary to this argument would be that the identification of potential protein ligands to IDA-Cu(I1) by ASA calculation with a 1.4A probe size is, in essence, erroneous. Structural analysis of a molecule on a computer graphics screen provides theoretical models of mechanisms that should ideally be tested further by means of genetic engineering techniques. The potential role of His-41 as an affinity ligand to IDA-Cu(I1) was thus explored by constructing the XI mutant, His-41 + Lys (H41K). The mutated protein was expressed in E. coli to also yield an active, soluble enzyme, and it could be purified by means of a traditional, multistep purification protocol that includes ammonium sulfate fractionation and hydrophobic, size-exclusion, and anion-

\A372

IN IMAC

21

exchange chromatography (see Fig. 1 of Ref. 19). An interesting observation was that H41K behaved indistinguishably from wild-type XI during this purification. Moreover, catalytic and stability properties remained similar to those of wild type (19). Since position 41 (Fig. 2) is surrounded by solvent, these findings lend support to the contention that the wild-type overall structure has been preserved in the mutant enzyme. Under these conditions, if we exclude the site of mutation, H41K should maintain an otherwise constant background and, therefore, constitute an unequivocal control for verifying the role ascribed to this precise electron-donor group in the IDA-Cu(I1) recognition mechanism. IDA-Cu(I1) IMAC of total cell lysate from E. coli expressing the mutant enzyme yields the chromatographic profile shown in Fig. 3A: Elimination of histidine at position 41 in XI leads to the complete disappearance of the absorbance peak eluting at 69 min, with no otherwise apparent alteration of the “background” absorbance trace. IDA-Cu(I1) IMAC of the H41K mutant, previously purified according to the traditional protocol described above, gives the chromatogram shown in Fig. 3B, where a single absorbance peak is shown to elute, unretained, in the flow-through at 8 min. Therefore, these results clearly establish that XI adsorption to IDA-Cu(I1) is totally abolished as a

A4CLv

A3LE A45-E 4

I

FIG. 2. Structure of wild-type D-xylose isomerase around histidine 41. Histidine 41 of subunit A is shown facing the solvent. Only side chains of interest are represented. His-41 and surrounding glutamates that may eventually participate in a second stage of metal-chelate coordination are shown in bold. Hydrogen atoms and main-chain oxygens have been omitted for the sake of simplicity. (Reprinted with permission from Ref. 19. Copyright 1992 American Chemical Society.)

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T. MRABET

result of the His-41 + Lys mutation. Furthermore, the participation of other putative, independent, electron donors in XI binding to the metal-chelate complex can clearly be ruled out, since retardation of the mutant on the chelating gel would have otherwise been observed. Therefore, the combination of protein X-ray structure analysis, molecular modeling, site-directed mutagenesis, and mutant characterization highlights an effective and unambiguous approach to proving the direct implication of a surface electron-donating residue in IMAC.

CONCLUDING REMARKS Our understanding of the mechanisms that govern selectivity in protein adsorption to metal-chelate complexes has been for the most part phenomenological. In this article, experimental procedures for IMAC are described and discussed in detail. Hence, some empirical rules of interactions between electron donors and chelated metals have been reviewed and further dissected to substantiate, but also on occasion to rectify, previous interpretations. This analysis is shown to form a rational framework for predicting protein behavior

in IMAC and, furthermore, provides new insights into how the composition of chromatographic solvents can influence protein adsorption to and desorption from immobilized metal-chelate complexes. On this basis, IDA-Cu(I1) IMAC has been optimized to allow the purification of recombinant A. missouriensis D-xylose isomerase in a single chromatographic step starting from crude cell lysates of the E. coli expression host. Moreover, a rather high degree of purification (>99%) could be achieved, even though the protein does not appear to exhibit a large affinity for the immobilized metal (see Fig. 1). It is worth stressing that IMAC is an exquisite approach to purifying engineered mutant proteins. In affinity-based separation, the chromatographic contact region on the surface of a protein is as small as a few hundred square angstroms (43). In IMAC, this contact area is likely to be even more reduced, given the small size of the immobilized ligand. Therefore, only structural changes that affect the amino acid composition in the contact region (e.g., H41+ K) will modify chromatographic behavior. Indeed, IDA-Cu(I1) IMAC has been reproducibly used for the rapid purification of many XI variants, some of which are quite unstable. A fundamental question in biology pertains to the structural and physicochemical bases for molecular recognition. This study shows that a multidisciplinary

A

1

0.5 -

0.4

-

0.3

-

0.2

-

0.1

-

0.0

-

I E z 8 3 e

2 0

40

60

80

100

120

$

_

14

Retention Time (min)

3. IDA-Cu(I1) IMAC of the H41K mutant of D-xylose isomerase. (A) Sample representing the mutant H41K (-11 mg). (B) An aliquot (2.5 mg) of H41K purified according to a previously (19; see text). Solvents and gradient program are those described in the legend to Fig. 1. (Reprinted 1992 American Chemical Society.)

FIG.

40

60

80

100

Retention Time (min)

total lysate from E. coli cells producing described traditional, multistep protocol with permission from Ref. 19. Copyright

MOLECULAR

RECOGNITION

approach offers an effective “protein engineering” means of precisely evaluating the contribution of electron-donating amino acid residues on a protein surface in terms of their participation in metal-chelate recognition. This has led to the demonstration that histidine 41 in A. missouriensis XI is the predominant ligand to IDA-Cu(I1). This approach further enhances our understanding of the features associated with the nature and accessibility of functional groups in proteins, as well as surface size, shape, and physicochemical characteristics, all of which can influence both specificity and binding affinity. Insights that simultaneously integrate physical, chemical, and structural aspects of protein/ligand interactions are likely to have profound implications in our evaluation of theoretical models for molecular recognition.

23

IN IMAC

11. Sulkowski, E. (1987) Protein Purification (UCLA Symposia on Molecular and Cellular Biology, New Series (Burgess, R., Ed.), Vol. 68, pp. 1499162, Liss, New York. 12. Corradini, W. (1988)

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D. D., Dempsey, B., and Serjeant, E. P. (1981) for Organic Acids and Bases, p. 61, Chapmann

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ACKNOWLEDGMENTS

21. Hegetschweiler,

K., and

Saltman,

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107-109. The experimental work described in this article was performed in the laboratories of Plant Genetic Systems, Gent, Belgium, and was part of a protein engineering project also supported by Gist brocades, Delft, The Netherlands, and Amylum, Aalst, Belgium. The excellent technical assistance of Ilse Van den brande and Annemie Van den Broeck is gratefully acknowledged. I thank Drs. Philippe Alard, Roland Gordon-Beresford, Philippe Delhaise, Michel Bardiaux, Philippe Berthet, Ignace Lasters, M. Vijayalakshmi, Eugene Sulkowski, and Shoshana Wodak for many enlightening discussions.

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