Bioconjugate Chem. 2006, 17, 300−308
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NMR and Crystallographic Characterization of Adventitious Borate Binding by Trypsin Thomas R. Transue, Scott A. Gabel, and Robert E. London* National Institute of Environmental Health Sciences, Laboratory of Structural Biology, Box 12233, Research Triangle Park, North Carolina 27709. Received July 25, 2005; Revised Manuscript Received November 23, 2005
Recent 11B NMR studies of the formation of ternary complexes of trypsin, borate, and S1-binding alcohols revealed evidence for an additional binding interaction external to the enzyme active site. We have explored this binding interaction as a prototypical interaction of borate and boronate ligands with residues on the protein surface. NMR studies of trypsin in which the active site is blocked with leupeptin or with the irreversible inhibitor 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF) indicate the existence of a low-affinity borate binding site with an apparent dissociation constant of 97 mM, measured at pH 8.0. Observation of a field-dependent dynamic frequency shift of the 11B resonance indicates that it corresponds to a complex for which ωτ . 1. The 0.12 ppm shift difference of the borate resonances measured at 11.75 and 7.05 T, corresponds to a quadrupole coupling constant of 260 kHz. A much larger 2.0 ppm shift is observed in the 11B NMR spectra of trypsin complexed with benzene boronic acid (BBA), leading to a calculated quadrupole coupling constant of 1.1 MHz for this complex. Crystallographic studies identify the second borate binding site as a serine-rich region on the surface of the molecule. Specifically, a complex obtained at pH 10.6 shows a borate ion covalently bonded to the hydroxyl oxygen atoms of Ser164 and Ser167, with additional stabilization coming from two hydrogen-bonding interactions. A similar structure, although with low occupancy (30%), is observed for a trypsin-BBA complex. In this case, the BBA is also observed in the active site, covalently bound in two different conformations to both His57 N and Ser195 Oγ. An analysis of pairwise hydroxyl oxygen distances was able to predict the secondary borate binding site in porcine trypsin, and this approach is potentially useful for prediction of borate binding sites on the surfaces of other proteins. However, the distances between the Ser164/Ser167 Oγ atoms in all of the reported trypsin crystal structures is significantly greater than the Oγ distances of 2.2 and 1.9 Å observed in the trypsin complexes with borate and BBA, respectively. Thus, the ability of the hydroxyl oxygens to adopt a sufficiently close orientation to allow bidentate ligation is a critical limit on the borate binding affinity of surface-accessible serine/threonine/ tyrosine residues.
INTRODUCTION Analysis of the human genome is expected to expand the number of potential protein drug targets by an order of magnitude (1). In contrast with enzymatic targets for which activity-based assays are generally straightforward, it can be anticipated that in many cases drug development will target the interruption of protein-protein interactions involved in signal transduction processes. Target sites on protein surfaces are not typically characterized by the recessed cleft topology of most enzyme active sites and thus exhibit a reduced tendency to trap small molecules that can serve as lead compounds for ligand development. This analysis is supported by recent NMR screening studies, which indicate that the ability of small molecules to bind noncovalently to proteins is strongly dependent on local topology, being largely determined by the existence of pockets with optimal volume, compactness, and surface roughness (2). Boric acid forms complexes with sugars and other small molecules containing hydroxyl, carboxyl, or ring nitrogen functionalities (3-7). The presence of these groups on proteins suggests that analogous protein complexes should exist. Consistent with this expectation, binding interactions between borate and cytochrome c have been detected by NMR spectroscopy (8, 9). Noncovalently associated boric acid (10; pdb code 1B33) and borate (11; pdb code 1A95) occasionally have been observed in protein crystal structures; however, we are not aware of previous reports of covalent complexes involving borate and the residues on protein surfaces. Of course, the binding of
boronate inhibitors to the active site of serine proteases has been studied extensively (12-23 and references therein). Recent 11B NMR studies of ternary complexes formed from trypsin, borate, and S1-binding alcohols revealed the existence of an additional borate binding site(s) of unknown location and composition (24, 25). The 11B NMR characteristics of this additional borate resonance are consistent with a polydentate, tetrahedral borate species that is in slow exchange with uncomplexed borate. The present studies were undertaken to further clarify whether this resonance corresponds to a secondary binding site on the trypsin or to a contaminant, to characterize the binding affinity and structure of the adduct formed, to identify the boron ligands involved in the complex, and to evaluate whether structurally related boronate molecules might also interact with the putative secondary binding site. It was further postulated that if borate does bind to a secondary binding site on the surface of trypsin, then this type of binding interaction is unlikely to be unique. The specificity of such interactions might lead to a different type of ligand selectivity than that which can be achieved based on a combination of noncovalent hydrophobic and electrostatic interactions.
EXPERIMENTAL PROCEDURES Crystallography. Type IX porcine pancreatic trypsin (Sigma, St. Louis, MO) was crystallized in the presence of 100 mM guanidino-3-propanol (G3P)1 as described previously (25). For complexes containing borate bound at the second site, a crystal
10.1021/bc0502210 Not subject to U.S. Copyright. Published 2006 by American Chemical Society Published on Web 02/23/2006
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Borate Binding by Trypsin Table 1. Crystallographic Data Summary data set pH soaked with cryoprotectant unit cell space group resolution (Å) no. of observations unique reflns Rsym (%) (last shell) I/(σI) (last shell) mosaicity (deg) completeness (%) (last shell) redundancy Rcryst (%)a Rfree (%)b no. of waters mean B value (Å) (protein only)
Crystallographic Data Statistics borate complex 10.6 200 mM borate, 50 mM G3P, 5 mM CaCl2 90% saturating MgSO4 a ) 76.30, b ) 53.45, c ) 46.47 Å R ) β ) γ ) 90° P212121 1.25 581 590 47 257 8.2 (31.0) 28.1 (4.7) 0.41-0.75 (avg ) 0.58) 88.6 (70.5) 12.3 Refinement Statistics 13.8 15.2 413 13.2 (10.9)
BBA complex 10.5 1 M BBA 1 M CAPS buffer a ) 76.85, b ) 53.70, c ) 46.71 Å R ) β ) γ ) 90° P212121 1.5 317 430 31 465 8.7 (44.6) 17.2 (2.1) 0.5 99.4 (96.0) 5.5 16.8 18.9 325 18.8 (16.4)
rms Deviation from Target Values bond length (Å) bond angle (deg)
0.011 2.14
0.008 2.02 Ramachandran Statistics
residues in favored (98%) regions (%) residues in allowed (>99.8%) regions (%) a
97.7
98.6
100.0
100.0
R-factor ) ∑||Fo| - |Fc||/∑|Fo| calculated from working data set. b Free R is calculated from 5% of data randomly chosen to be excluded in refinement.
was transferred in four 1 min steps to a final solution containing 90% saturated MgSO4 (acting as a cryoprotectant), 200 mM borate, pH 10.6, 50 mM G3P, and 5 mM CaCl2. The solutions for each step contained ratios of mother liquor to final solution of 50:50, 10:90, 0:100, and 0:100. The crystal was then flash frozen in liquid nitrogen and mounted for data collection. For benzene boronic acid (BBA), a crystal was transferred via several dilutions with mother liquor to a final solution containing 1 M CAPS buffer pH 10.5 (acting as a buffer and cryoprotectant) and 1 M BBA. The crystal was then frozen in liquid nitrogen and mounted for data collection. Data were collected as described previously (25) using a Rigaku rotating anode generator and an R-AXIS IV detector (Molecular Structure Corporation). Data were reduced using DENZO and SCALEPACK (HKL Research) (26). All models were refined using a maximum likelihood target against all intensities using CNS (27). Force parameters were taken from the AMBER force field (28). The relative occupancy of alternate models (including alternate side chain conformations and alternately occupied borate or BBA binding sites) was iteratively estimated by minimizing difference electron density and by comparing the refined B-factors looking for a minimal difference. Water molecule occupancies were refined and then examined manually to avoid very low occupancies or very high B factors. For the borate crystal, a complete data set to 1.25 Å resolution was collected. The BBA complex crystal produced a data set complete to 1.5 Å. Data collection statistics as well as refinement statistics are provided in Table 1. Crystal structures showing the complex of trypsin with borate and with BBA have been deposited in the protein data bank with accession numbers 2A31 and 2A32, respectively. 1 Abbreviations: 4AB, 4-amino-1-butanol; AEBSF, 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride; AEBS-trypsin, AEBSFmodified trypsin; BBA, benzene boronic acid (sometimes referred to as phenylboronic acid); CAPS, N-cyclohexyl-3-aminopropanesulfonic acid; G3P, guanidino-3-propanol.
NMR Spectroscopy. 11B NMR spectra were obtained on a Varian INOVA 500 NMR spectrometer operating at 160.6 MHz for boron or on a Varian Gemini 300 NMR spectrometer operating at 96.3 MHz. In both cases, the 11B measurements were performed using quartz NMR tubes (Wilmad, Buena, NJ) and 5 mm variable-temperature broadband probes that had been modified to reduce the 11B background signal. The probe used for the 11B studies on the Gemini 300 was modified by JS Reasearch (Quincy, MA), and the probe used for the 11B studies on the INOVA 500 was modified by Alex Funk. For the line width analysis and for determination of the apparent dissociation constant of the trypsin-borate complex involving the second binding site, the trypsin was pretreated with 5 mM 4-(2aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF) for 1 h at 4 °C to irreversibly block binding at the active site. We have referred to trypsin modified in this way as AEBStrypsin. Additionally, 3 mM leupeptin (Bachem, King of Prussia, PA) was added to the sample to competitively bind to any unmodified active sites. Typical INOVA 500 spectral parameters were as follows: sweep width, 16 kHz; acquisition time, 1.0 s; transients, 2000. Gemini 300 spectral parameters were as follows: sweep width, 10 kHz; acquisition time, 0.6 s; transients, 44 400. 11B shifts are referenced to external boric acid at pH 4.0. Since the boric acid/borate concentrations in most of the studies were relatively high, the spectra shown correspond to an expanded region around the bound borate species and do not include the resonance of the uncomplexed boric acid/borate. In most cases, the curved baseline resulting from the large boric acid/borate resonance was corrected using the polynomical fit of the Varian VNMR software. Software. We developed a simple PERL script to check for neighboring hydroxyl oxygen atoms in any given PDB file. The script searches for serine, threonine, and tyrosine residues, records the coordinates of the hydroxyl oxygen atoms, and then computes pairwise distances. The residues, atoms, and distance
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Figure 1. Effect of the leupeptin inhibitor on the binding of borate: (A) 11B NMR spectrum of 2 mM trypsin, 50 mM borate, and 10 mM 4-aminobutanol in 50 mM HEPES, pH 8.0; (B) 11B NMR spectrum of the sample shown in panel A with the addition of 3 mM leupeptin. 11B spectra were obtained at 160.6 MHz on a Varian INOVA 500 at 25 °C.
for any pair closer than a defined distance (set to 6.0 Å in this case) are tabulated.
RESULTS NMR Studies of the Trypsin-Borate Complex. Recent NMR studies of trypsin-borate complexes yielded evidence for additional borate binding interactions extending beyond the active site of the enzyme (24, 25). For example, 11B NMR studies reveal a resonance that can be assigned to a ternary trypsin-borate-4-aminobutanol (4AB) complex that forms at the active site in the presence of 5 mM borate. However, further increases in the borate concentration resulted in increased intensity of this resonance, which in some cases exhibited a structure indicating that it was composed of more than one component peak (Figure 1A). The presence of an additional binding site was confirmed by the addition of leupeptin, an inhibitor expected to compete with the active site borate-4AB complex (Figure 1B). Since borate binds to a large number of small molecule ligands that might be present in the enzyme preparation, for example, glycerol, citrate, tris buffer, etc., it was initially unclear whether this additional resonance resulted from an interaction with trypsin or with small molecule contaminants. However, 1H NMR studies of the trypsin-borate complex failed to provide evidence of significant small molecule contaminants. Additional dialysis of the trypsin against buffer using a Slide-A-Lyzer also failed to eliminate the additional borate signal. Since protein surfaces contain the same functional groups present in many small borate ligands, it was hypothesized that low-affinity borate binding sites might be a general characteristic of protein surfaces. NMR evidence for the existence of such sites in cytochrome c has previously been reported (8, 9). Exploration of the surface of the trypsin molecule immediately revealed a serine-rich region on the surface including residues Ser164, Ser166, Ser167, Ser170, and Ser171 as a potential borate binding site. To obtain a more quantitative estimate of the binding affinity, the trypsin was treated with AEBSF, an irreversible, activesite-directed inhibitor of the enzyme, to eliminate this binding interaction. Boron-11 NMR studies were then performed on a sample containing 2 mM AEBS-trypsin, 2 mM lactate, and 50 mM HEPES buffer, pH 8.0. The sample also contained 3 mM leupeptin to further suppress any borate complex formation with unreacted trypsin. Lactate, which forms a binary lactate-
Transue et al.
Figure 2. Borate titration of trypsin. 11B NMR spectra of a sample containing 2.0 mM lactate, 2.0 mM AEBS-trypsin, 3 mM leupeptin, and 50 mM HEPES, pH 8.0, as a function of borate concentration. The identity of the upfield resonance at δ ) -19.5 ppm, which becomes apparent at high borate concentration, is tentatively assigned to pentaborate (4). 11B spectra were obtained at 160.6 MHz on a Varian INOVA 500 at 22 °C.
borate complex (3), was used as an internal standard. As can be seen from Figure 2, the 11B NMR spectrum shows the progressive formation of the borate-lactate and (AEBStrypsin)-borate complexes, where the latter must arise from borate binding to a second site since the active site has been blocked. An additional resonance at δ ) -19.5 ppm apparent at higher borate concentrations, is assigned to the borate component of pentaborate, based on the shift data reported by Coddington and Taylor (4). Conceivably, the pentaborate or other polyborate species present at high borate concentrations could also bind to trypsin. Based on the data of Pizer and Selzer (3), the lactate-borate complex has an apparent KD value of 128 mM at pH 8. To analyze the data of Figure 2, we also need to account for the fact that we are presumably only observing the central 1/2 T -1/2 transition for the borate-trypsin complex, corresponding to a fractional peak intensity given by (29)
I1/2,-1/2 )
(
)
3 2s + 1 ) 0.4 for 2 2s(2s + 2)
11
B
(1)
where I1/2,-1/2 is the fractional intensity of the central transition for a quadrupolar nucleus with half-integral spin value s with the latter equality corresponding to 11B for which s ) 3/2. Analysis of the data shown in Figure 2 gave a value of KD ) 97 mM for the (AEBS-trypsin)-borate complex. This interaction is similar to the inhibition constant of 80 mM measured previously for borate inhibition of trypsin (24). For reference, the apparent KI for the inhibition of trypsin by borate is lowered to 18 mM in the presence of saturating 4-aminobutanol, which binds cooperatively (24). Crystal Structure of the Borate Complex. Previous crystallographic studies have verified the formation of ternary and quaternary trypsin-borate-alcohol complexes that form in the active site (25, 30), confirming the results of earlier NMR studies (24). As discussed previously, the nature of these complexes is also apparently influenced by crystal packing effects (25, 30). No additional borate binding interactions were observed in crystallographic studies performed at pH 8.0 (e.g., Figure 3a). To obtain more specific insight into the second borate binding site on trypsin, a crystal containing a trypsin-G3P complex
Borate Binding by Trypsin
Figure 3. Crystallographic structure of the second borate binding site in trypsin: (a) X-ray structure of a serine-rich region of the trypsin surface obtained at pH 8.0 in the presence of 200 mM borate. PDB code: 1S6F; (b) structure of the same region of the protein crystallized at pH 10.6 in the presence of 200 mM borate, showing a borate ion covalently bound to the hydroxyl oxygens of Ser164 and Ser167; (c) covalent and hydrogen bonding interactions of borate bound at site 2. For clarity, the boron is indicated in green and the β-carbons in yellow. The two hydrogen bonding interactions involving the borate anion are indicated by large dotted lines. Small dotted lines indicate hydrogen bonds characteristic of R-helical secondary structure.
was soaked in a solution containing 200 mM borate at pH 10.6, and the resulting complex was analyzed by X-ray crystallography (Table 1). In this crystal form, a lattice contact inhibits ternary complex formation at the active site (25, 30). However, the structure reveals a non-active-site bidentate borate complex with an occupancy of 50% formed from the Ser164 and Ser167 hydroxyl groups (Figure 3b). In addition to the covalent interactions with Ser164 and Ser167 hydroxyl oxygens, the complex is stabilized by a pair of hydrogen bonds (Figure 3c): the amide nitrogen of Ser164 is within hydrogen bonding range
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(2.8 Å) of one of the borate hydroxyl oxygens, and the amide nitrogen of Ser167 is within hydrogen bonding range (2.9 Å) of the covalently linked Ser164 Oγ. Electron density of the five electron boron atom is visible and continuous with that of the two serine oxygens, which are 2.2 Å apart. In the uncomplexed model representing the other 50% occupancy, the two serine hydroxyl oxygens are separated by 2.6 Å, and a water molecule is present at one of the sites occupied by a borate hydroxyl in the structure of the complex. At 1.25 Å resolution, the unbiased electron density (Fo - Fc before modeling borate), as well as the final 2Fo - Fc density, unambiguously identifies this site as forming two covalent linkages to borate. Crystal Structure of the Benzene Boronic Acid-Trypsin Complex. To further explore the borate binding site on the surface of trypsin that was identified by NMR and X-ray crystallography, we soaked the trypsin crystals in solutions containing a series of boronate ligands. In general, site 2 was found to be a relatively poor boronate binding site, presumably as a result of the lack of other useful interacting groups well positioned to stabilize the boronate complexes and, in the crystalline state, the potential limiting effects of crystal packing interactions. A complex of the enzyme with benzene boronic acid (BBA) bound to both the active and secondary sites was obtained at high pH using high BBA concentrations (Table 1). In this structure, BBA binds to the active site in either of two conformations, both of which involve bidentate covalent ligation to the His57 N and the Ser195 Oγ (Figure 4a). In one of the conformations, BBA(2), the phenyl ring extends into the S1 pocket and makes contact with the CR and Cβ of Gln192, while in the second, BBA(1), it extends away from the pocket toward Phe41 and the Cys42-Cys58 disulfide bond. In this binding mode, the orientation of the BBA phenyl ring more closely approximates the P1′ residue (after the cleavage site) and the interactions are with trypsin residues in the S1′ subsite (e.g., see ref 31). Binding to both the His N2 and the Ser Oγ has also been observed in some boronate complexes of γ-chymotrypsin (19). The BBA is also bound to the second borate-binding site identified above in studies with borate, but with a 30% occupancy (Figure 4b). As with the borate complex (Figure 3b,c), the BBA molecule forms two covalent bonds with the serine hydroxyl groups, and two of the boron-bound oxygen atoms are within H-bonding distance of Ser164 NH (2.9 Å) and Ser167 NH (3.0 Å). The phenyl ring extends away from the surface of the protein so that the hydrogen bond from the Ser164 amide nitrogen to the only free hydroxyl is the same as that seen in the borate complex. Electron density does not support the alternate orientation of the phenyl ring, presumably because this would result in an amide nitrogen to aromatic carbon clash. There was apparently some borate present in the BBA sample used, because borate was alternately observed in site 2, also with 30% occupancy (i.e., the phenyl ring has lower occupancy than the other three heavy atoms). The remaining 40% appears to contain unbound serine 164 and 167, rotated slightly to place the Oγ atoms about 2.8 Å apart. The observation of equal populations of trypsin-complexed BBA and borate in this study was somewhat surprising, since the borate was present only as a contaminant. Based on a measurement of the resonance intensities in the 11B NMR spectrum of the BBA, the borate contamination is ∼3%. BBA generally is expected to form tighter complexes due to the higher affinity for hydroxyl groups reflected in the lower pK value. An examination of lattice contacts suggests that unfavorable crystal packing effects may destabilize the trypsin-BBA complex. Specifically, the phenyl ring of the BBA comes in close proximity with the Ser61 and Arg62 residues of a symmetry-related molecule, which may exert a destabilizing effect. Alternatively, favorable interactions
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Figure 4. Crystal structure of the benzene boronic acid-trypsin complex: (a) Wall-eyed stereoview of the active site of the enzyme showing two similarly populated orientations of bound BBA; (b) interaction of BBA with the second borate bonding site formed from serine residues 164 and 167.
of borate with both of these residues might act to stabilize the borate complex. Of course, such interactions would not be present in solution. The Ser164/Ser167 Oγ-Oγ distances of 1.9 Å and 2.2 Å in the BBA and borate complexes, respectively, are significantly shorter than the distances observed for uncomplexed trypsin (see below). The need to force the Oγ nuclei together to achieve binding is thus probably an important constraint on the surface binding interactions observed here. Analysis of Dynamic Frequency Shifts. To further characterize the borate/boronate binding interactions with trypsin, we performed field-dependent 11B NMR studies on the complexes. In the slow tumbling limit, which characterizes the trypsinborate complexes under study here, quadrupolar nuclei with halfintegral spin, such as 11B, exhibit unusual relaxation and shift characteristics, which can be useful for understanding the nature of the complex. In this limit, the J(0) spectral density term, which typically makes the dominant contribution to transverse relaxation behavior in the slow tumbling limit, does not contribute to the relaxation of the central 1/2 T -1/2 transition, resulting in a relatively narrow signal, while the remaining transitions are very broad and generally unobserved (29, 3234). The central transition for the 11B in this slow tumbling limit is subject to a second-order dynamic frequency shift given by (34)
δ)
8s(s + 1) - 6 s2(2s - 1)2
(LQ(ω) - LQ(2ω))
(2)
LQ(ω) )
(
2
( )
)
3χ ωτ 160 1 + ω2τ2
where χ, the quadrupolar coupling constant, is equal to (h/(2π)), and τ is the isotropic rotational correlation time. In the limit ωτ . 1, we then obtain
( )( )
4s(s + 1) - 3 3χ2 1 χ2 3 ≈ for s ) 2 2 160 ω 40ω 2 s (2s - 1)
(4)
This can be expressed in ppm as (35)
δppm ≈
(6)
(3) e2Qq/
δ≈
Boron-11 NMR spectra for solutions containing 2 mM trypsin, 6 mM lactate, 250 mM 4AB, and 50 mM borate in 50 mM HEPES, pH 8.0, were obtained at both 7 and 11.75 T to evaluate the relaxation behavior of the 11B nucleus (Figure 5). The lactate standard was included to obtain a signal from a small molecule complex, which would not be subject to slow tumbling and should exhibit negligible dynamic frequency shifts. The 11B NMR spectra show, in addition to the free boric acid resonance, a resonance corresponding to the lactate-borate complex at -13.0 ppm and two poorly resolved peaks at -17.1 ppm, which we assign to the ternary trypsin-4AB-borate complex at the active site and to the binary trypsin-borate complex that involves complex formation with Ser164 and Ser167 discussed above (Figure 3). We note that the resolution of the two component peaks in this study is somewhat poorer than that in Figure 1, apparently due to a small drop in pH of the sample, as well as to the higher 4AB concentration used. Although the field-dependent shift difference is small, using the borate-lactate complex as a reference, both of the components of the trypsinborate complex shift downfield by 0.12 ppm at the higher magnetic field strength. Inserting the observed shift difference of 0.12 ppm into eq 5 and taking the ratio at the two fields gives
δ500 δ500 300 2 ) ) ) 0.36 δ300 δ500 + 0.12 500
where 2
shift. The second equality above is useful if the quadrupole coupling constant is expressed in hertz rather than radians per second.
( )
-2.5 × 104χ2 -2.5 × 104 e2Qq ) h ω2 ν02
2
(5)
where we have introduced the negative sign indicating an upfield
where we have assumed that χ is field-independent, giving for the dynamic frequency shift at 500 MHz, δ500 ) 0.0675 ppm. Based on this value, solution of eq 5 then gives χ ) 264 kHz. This small value is similar to typical values for deuterium and reflects the relative symmetry of the boron electronic structure in the bound borate. To further probe these effects, similar field-dependent 11B NMR studies were performed on a sample containing 2 mM trypsin, 6 mM lactate, 50 mM BBA, and 250 mM 4AB in 50 mM HEPES, pH 7.0. The lower pH was used in this case to provide better separation between the resonances of the free benzene boronic acid and the complexes, since this compound has a lower pK (8.72) than boric acid (8.98) (36). In contrast with the results for the borate complexes, the trypsin-BBA peaks, which cannot be individually resolved in this case,
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Borate Binding by Trypsin
Figure 5. Frequency dependence of the 11B NMR spectra of a trypsinborate complex: (A) spectrum obtained at 7 T (11B frequency ) 96.326 MHz); (B) spectrum obtained at 11.75 T (11B frequency 160.606 MHz). The sample contained 2.0 mM trypsin, 6.0 mM lactate, 50 mM borate, and 250 mM 4-aminobutanol in 50 mM HEPES, pH 8.0, buffer. Spectra were run at 25 °C.
Figure 6. 11B NMR spectra of a sample containing 6.0 mM lactate, 2.0 mM trypsin, 250 mM 4-aminobutanol, and 50 mM BBA in 50 mM HEPES, pH 7.0, buffer: (A) spectrum obtained at 7 T (11B frequency ) 96.326 MHz; (B) spectrum obtained at 11.75 T (11B frequency 160.606 MHz). Spectra were run at 25 °C. There is an apparent dynamic frequency shift of 2.0 ppm.
experience a significantly greater upfield shift at the lower field (Figure 6). On the basis of the observed shift difference of 2.0 ppm and using eq 5, we have
δ500 δ500 300 2 ) ) ) 0.36 δ300 δ500 + 2.0 500
( )
(7)
giving for the dynamic frequency shift at 500 MHz, δ500 ) 1.125 ppm. Insertion of this value into eq 5 gives χ ) 1.08 MHz for the quadrupole coupling constant of 11B in the benzene boronate complex with trypsin. This value is similar to the 0.87 MHz determined from spin lattice relaxation measurements of BBA in the presence of chymotrypsin (13). Of course, as noted above, this value actually corresponds to a mixture of species. However, it is presumably dominated by the local environment of the boron and hence not strongly dependent on the detailed differences of the various component complexes. We note that if there were other small molecule complexes present in this system besides the borate-lactate complex that was specifically introduced, the field-dependent data should allow resolution of these species. The absence of additional resonances, particularly at the lower field, is consistent with our previous conclusion that the additional binding interaction observed in our previous studies (24, 25) arose from binding to the second Ser164/Ser167
Figure 7. Temperature dependence of the 11B NMR spectrum of the BBA-trypsin complex. To eliminate the contributions from BBA bound to the active site, the trypsin was treated with AEBSF, as described in the methods section. The sample contained 2 mM AEBS-trypsin, 2 mM lactate, and 50 mM benzene boronic acid in 50 mM HEPES, pH 8.0. 11B NMR spectra were obtained at (A) 5, (B) 15, and (C) 25 °C.
binding site, rather than from binding to a small molecule contaminant. To obtain further insight into this relaxation behavior, analogous studies were performed on benzene boronic acid in the presence of another serine protease, subtilisin Carlsberg. Benzene boronic acid (13) and its 4-fluoro analogue (37) have been shown to bind to this protease. Interestingly, the measured dynamic frequency shift was found to be 2.0 ppm, essentially identical to the result for trypsin with its multiple binding sites (data not shown). This result is consistent with eq 5, which indicates that the only significant parameter in predicting this shift under the slow tumbling approximation is the quadrupole coupling constant, which is presumably very similar for the benzene boronate, whether it is complexed with trypsin or subtilisin Carlsberg. Line Width Effects in the BBA Complex. Temperaturedependent 11B NMR spectra of the (AEBSF-treated trypsin)BBA complex are shown in Figure 7. As in the above study, the sample also contained lactate (2 mM) to provide a reference borate complex. When the same set of approximations is used to analyze the relaxation behavior of the central component of the 11B resonance, the line width of the central transition is given by (29, 34)
∆ν1/2 )
1 2s + 3 {2JQ(ω) + ) πT2 πs2(2s - 1) 1 [(s + 3/2)(s - 1/2) - 1](JQ2 (2ω)} (8)
where
JQ(ω) )
(
3χ2 τ 160 1 + ω2τ2
)
(9)
where χ is the quadrupole coupling constant, e2Qq/(h/(2π)), nucleus S has spin s and Larmor frequency ω, τ is the rotational correlation time, and isotropic motion is assumed. In
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the limit ωτ . 1, we obtain
1 ∆ν1/2 ) ≈ πT2 2(2s + 3)
(
)(
Table 2. Hydroxyl Oxygen Distances in Crystal Structures
)
1 3χ2 1 3 1 1 + / )(s / ) 1) ) ((s + 2 2 8 160 ω2τ πs2(2s - 1) χ2 1 1 e2Qq 2 1 3 ) for s ) (10) 16π ω2τ 16π h ν 2τ 2 0
( )
where the second equality converts the quadrupole coupling constant to units of hertz. This inverse dependence on τ contrasts with the line width predictions based on other relaxation mechanisms such as the dipolar interaction and chemical shift anisotropy, which predict ∆ν1/2 ≈ J(0) ≈ τ. However, chemical exchange between uncomplexed and variously complexed forms of the borate ion also can make significant contributions to the line width. 11B line widths measured for BBA complexed with AEBSF-treated trypsin to eliminate the contribution of BBA bound to the active site were measured at 5, 15, and 25 °C and yielded values of 51.5, 62.9, and 82.4 Hz, respectively. We have compared this temperature dependence with the behavior predicted from an isotropic tumbling model:
η 4 τ ) πr3 3 kT
(11)
where η is the viscosity of the solution. Using a table of water viscosity from the Handbook of Chemistry and Physics (38), the ratio of correlation times at the 5, 15, and 25 °C is calculated to be 1.0:0.74:0.55, so that based on eq 10 above, the ratios of the line widths at the three temperatures are predicted to be 1:1.35:1.82. The three experimental line widths given above have the ratio 1:1.22:1.60, in reasonable agreement with the prediction. Using eq 10, the rotational correlation times calculated from the line width of the central component of the 11B signal at 5, 15, and 25 °C are 1.74 × 10-8, 1.44 × 10-8, and 1.10 × 10-8 s, respectively. These values are reasonable for a protein the size of trypsin (MW ) 23 281). Prediction of Adventitious Borate Binding Sites. Studies of model borate complexes indicate that borate readily interacts with hydroxyl, carboxyl, ring nitrogen, and occasionally amino groups. Typically, multidentate chelation is sufficient to give rise to slowly exchanging 11B resonances, although at sufficiently high fields and low temperatures, even monodentate interactions can sometimes produce resolved resonances. Despite the ubiquitous occurrence of these potential ligands in proteins, the present NMR and crystallographic studies detected only one nonspecific binding site at borate concentrations under 100 mM involving a pair of serine hydroxyl groups. Since the multidentate complexes formed with hydroxyl ions are usually sufficiently stable to allow the observation of 11B resonances that are in slow exchange (e.g., ref 4), these are generally the easiest to observe. Based on the hypothesis that multidentate interactions with hydroxyl ligands provide the most prevalent basis for nonspecific borate binding, a calculation of the hydroxyl distances should provide a crude predictive tool for potential borate binding sites. Results of this analysis are summarized in Table 2. The closest pairing of hydroxyl groups occurs for the hydrogen-bonded S190 and Y228 residues that are buried in the interior of the protein and hence not accessible for binding. The S164-S167 site identified in the present study corresponded to the second shortest hydroxyl-hydroxyl distance, supporting the conclusion that this is a valid approach for predicting adventitious borate binding. Based on the results summarized in Table 2, we examined a sample containing
residue 1
residue 2
S190 S164 S164 S37 T144 S146 S214 T125 S109 S130 S166 S170 S149 S166 Y172 Y94 Y29 Y59 T177
Y228 S167 S166 Y39 S150 S147 T229 S127 S110 T134 S170 S171 Y151 S167 S217 S96 S139 S61 S178
distancesa min (mean)
distancesb min (mean)
2.74 (2.77) 2.80 (3.42) 3.03 (4.43)
2.70 (2.83) 2.92 (3.26) 3.36 (3.65) 3.20 (3.43) 3.97 (4.06) 3.50 (4.78) 3.78 (3.86) 4.00 (4.23)
3.32 (3.69) 4.19 (5.19) 3.81 (3.98) 4.14 (4.78) 4.28 (5.81) 4.40 (5.20) 4.44 (5.88) 4.45 (5.05) 4.89 (4.94) 4.89 (5.80)
4.18 (4.82) 4.90 (6.18) 4.75 (5.66) 5.18 (5.39) 4.56 (5.15) 4.81 (4.93) 5.00 (5.05) 5.01 (5.22) 4.96 (5.21)
a For porcine trypsin crystal structures 1S83, 1FNI, 1MCT, 1QQU, and 1AVW. b For bovine trypsin crystal structures 1HJ9, 1UTN, 1J8A, 1O3D, and 5PTP.
bovine trypsin plus 50 mM borate under conditions similar to those of Figure 1 and observed a borate resonance with a shift similar to that of the bound borate in the procine trypsin-borate complex (spectrum not shown). Thus, it is likely that a similar complex forms with bovine trypsin. The Ser164-Ser167 Oγ-Oγ distance was determined to be 2.2 Å in the borate complex and 1.9 Å in the BBA complex. In contrast, the Ser164/Ser167 Oγ-Oγ distances in the crystal structures of uncomplexed trypsin are significantly longer so that a conformational change must occur upon borate/boronate complex formation. Depending on the protein, this geometric constraint may not be achievable. A survey of a series of proteins currently under study in our lab in the presence of 50 mM borate generally did not show a significant 11B resonance corresponding to bound borate. The protein ubiquitin provides a good example of a protein containing a pair of hydroxyl groups, corresponding to Thr7 and Thr9, which appear to provide a potential borate binding site. Analysis of three high-resolution structures, 1UBQ, 1UBI, and 1D3Z, gave a mean Oγ-Oγ distance of 3.18 Å for Thr7/Thr9, shorter than the mean Oγ-Oγ distance for Ser64/ Ser167 in trypsin, but at 50 mM borate, no bound resonance was observed. Analogous to the trypsin structure, the Oγ of Thr7 appears to be H-bonded to the Thr9 NH, orienting the side chain toward the Thr7 Oγ. In this case, variation of the χ1 angles for both threonine residues to achieve closest approach of the Oγ atoms reduces the Oγ-Oγ distance to ∼2.6-2.7 Å, substantially greater than the distance of 2.2 Å for Ser164Ser167 in the trypsin-borate complex, and achieving the 2.6 Å separation leads to significant steric conflict. Hence, the inability to reach sufficient proximity for bidentate ligation may be a limiting factor for borate complexation with Thr7/Thr9 in ubiquitin.
CONCLUSIONS The complexes formed by boronate ligands with serine proteases and mechanistically related enzymes have been characterized extensively by X-ray crystallography, NMR spectroscopy, and other physical methods (12-23). These complexes generally contain a covalent bond between the active site serine Oγ and the boron to form a boronate structure believed to mimic the transition state, although binding to the active site histidine imidazole N2 has also been observed (15, 16, 19). Structurally analogous ternary and quaternary complexes involving serine proteases, borate, and alcohols also have been
Borate Binding by Trypsin
observed recently (24, 25, 30). Recent 11B NMR studies of trypsin complexes also provided evidence that at higher concentrations, an additional interaction between borate and trypsin was occurring (24, 25). The studies described here were undertaken to more fully characterize this adventitious binding with a view toward exploitation of such interactions for the more general development of surface-directed protein ligands. In the present studies, we have determined that the secondary trypsin-borate binding site involves covalent binding to the hydroxyl groups of Ser164 and Ser167, with additional stabilization resulting from a pair of hydrogen bonding interactions. An apparent borate dissociation constant of 97 mM at pH 8.0 was determined for this site, which can also be occupied by benzene boronic acid. Since the interaction does not involve residues of the serine protease active site, it is likely that this type of binding interaction is not unique. An upfield borate resonance with characteristics similar to the bound borate was also observed in a sample of bovine trypsin. Interesingly, the KD value of 97 mM is similar to the KI value for borate for the active site of trypsin (24) and other serine proteases (39). In contrast with the surface binding site, active site binding is typically monodentate (however, note Figure 4a) and is characterized by much higher kon and koff rates, so no separate 11B resonance can be observed. In the presence of alcohols that form a ternary complex, a bidentate borate interaction with the enzyme and the alcohol creates a more stable complex so that a separate 11B resonance does become observable (24, 25). It was initially anticipated that if borate binding to a second site located on the protein surface was occurring, then it would be possible to identify a boronate compound with greater affinity for this site due to the contribution of additional interactions with the substituent on the boron. However, none of the boronate compounds studied showed evidence of significantly enhanced affinity. One explanation for this observation is the significantly shorter Oγ-Oγ distance between the ligating Ser164/Ser167 hydroxyl oxygens observed in the BBA compared with the borate complex. Since these Oγ-Oγ distances are both substantially shorter than the values observed in the absence of borate/BBA (Table 2), it appears that in both cases the site must adjust for bidentate ligation, and the adjustment must be greater in the case of BBA. A second explanation suggested by the structure of the trypsin-BBA complex is that the phenyl ring of the BBA is approximately normal to the protein surface, precluding any additional interaction with the protein. Since serine residues contribute two of the oxygen ligands and a third hydroxyl group is hydrogen bonded to the Ser164 NH group, the carbon substituent of the boronate is forced to point away from the surface of the protein, so additional interactions of the boronate ligand with the protein in site 2 are essentially eliminated. In this orientation, unfavorable lattice contact may further destabilize the BBA complex, stabilize the borate complex, or both. The results of the present study pose an interesting question: what properties of binding site 2 confer the borate binding affinity, and how unique is this type of interaction? Studies of small molecule complexes reveal that optimal positioning of boron ligands is essential for obtaining high binding affinity. For example, a study of the borate complexes formed by a series of pentoses demonstrated that borate complexes are formed exclusively with the cis-hydroxyl groups of the furanose and not the pyranose forms of the sugars (40). To form a borate binding site on the protein surface with reasonable affinity, it appears critical to have at least two boron ligands that are positioned to allow bidentate ligation. Hydroxyl groups that are not well positioned or that are highly dynamic and rapidly sample many different conformational states will not be optimal for borate binding. From this perspective, we note that the
Bioconjugate Chem., Vol. 17, No. 2, 2006 307
assigned hydrogen bonding interaction between Ser164 Oγ and the Ser167 NH group that characterizes the trypsin complexes with both borate and BBA appears to be present in a majority of the uncomplexed structures as well. This interaction probably contributes to borate binding by orienting the Ser164 side chain toward Ser167. Alternatively, in trypsin crystals with no borate bound to the second site, the side chain of Ser167 typically adopts multiple conformations, some oriented so that the OγH is within H-bonding range of the Ser167 carbonyl oxygen and some oriented with OγH within H-bonding distance of the Ser164 side chain. These results are consistent with a moderate affinity borate binding site and further suggest that a higher affinity borate binding site characterized by more optimally positioned and more conformationally constrained hydroxyl groups could, in principle, exist in other proteins. Most critically, the pair of Oγ ligands contributed by serine, threonine, or tyrosine must be able to move close enough to form a bidentate complex. Additional stabilization of the trypsin complexes with both borate and BBA results from a hydrogen bond between one of the borate hydroxyl oxygens and the Ser164 NH (Figures 3 and 4b). Since this NH group is on the same residue involved in borate chelation, it is possible that this type of interaction could be a typical feature of other surface binding sites. A positively charged residue, functionally analogous to the oxyanion hole in the active site of a serine protease (41), could also provide additional stability. There appears to be no well-positioned cationic residue that could further stabilize the bound borate at site 2 of trypsin; however, proximity to the N-terminus of the R-helix with its associated dipole moment may make the Ser164/ Ser167 binding site more favorable for anionic species. Since the secondary borate binding site on the surface of trypsin identified in this study does not involve catalytic residues, it is unlikely that this type of binding interaction is unique to trypsin. Potentially, this type of borate binding interaction could form the basis for the development of boronate ligands that target the protein surface. A simple calculation of pairwise hydroxyl oxygen distances provides a rough predictive tool of potential borate binding sites, particularly if inaccessible sites are excluded. However, as illustrated by the lack of significant binding to ubiquitin, it must be possible to easily reorient the hydroxyl oxygens to within ∼2.5 Å, or perhaps slightly longer distances in some cases, without significantly perturbing the local protein structure to form a bidentate complex. The present results also illustrate the potential of dynamic frequency shifts of boronate ligands to distinguish ligands on the basis of rotational correlation times and hence molecular weight. This is a significant issue for borate and boronate compounds due to potential binding interactions with many small molecules. Additionally, this analysis provides a useful approach for the determination of quadrupole coupling constants and for obtaining dynamic information related to the protein-borate/boronate complex.
ACKNOWLEDGMENT The authors are grateful to Dr. Lawrence Werbelow and Dr. Eugene DeRose for suggestions and helpful discussions. This research was supported by the Intramural Research Program of the NIH, and NIEHS.
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