In Crystals of Complexes of Streptavidin with Peptide Ligands Containing the HPQ Sequence the pK a of the Peptide Histidine Is Less than 3.0*

The pH dependences of the affinities for streptavidin of linear and cyclic peptide ligands containing the HPQ sequence discovered by phage display were determined by plasmon resonance measurements. At pH values ranging from 3.0 to 9.0, theK d values for Ac-AEFSHPQNTIEGRK-NH2, cyclo-Ac-AE[CHPQGPPC]IEGRK-NH2, and cyclo-Ac-AE[CHPQFC]IEGRK-NH2, were determined by competition, and those for cyclo-[5-S-valeramide-HPQGPPC]K-NH2 were determined directly by equilibrium affinity measurements. TheK d values of the ligands increase by an average factor of 3.0 ± 0.8 per decrease in pH unit between pH ∼4.5 and pH ∼6.3. Below pH ∼4.5 there is a smaller increase inK d values, and above pH ∼6.3 theK d values become relatively pH-independent. We determined the crystal structures of complexes of streptavidin with cyclo-[5-S-valeramide-HPQGPPC]K-NH2 at pH 1.5, 2.5, 3.0, and 3.5, with cyclo-Ac-[CHPQFC]-NH2 at pH 2.0, 3.0, 3.6, 4.2, 4.8, and 11.8, with cyclo-Ac-[CHPQGPPC]-NH2 at pH 2.5, 2.9, and 3.7, and with FSHPQNT at pH 4.0 and compared the structures with one another and with those previously determined at other pH values. At pH values from 3.0 to 11.8, the electron density for the peptide His side chain is strong, flat, and well defined. A hydrogen bond between the Nδ1 atom of the His and the peptide Gln amide group indicates the His of the bound peptide in the crystals is uncharged at pH ≥ 3.0. By determining selected structures in two different space groups, I222 with two crystallographically inequivalent ligand sites and I4122 with one site, we show that below pH ∼3.0, the pK a of the bound peptide His in the crystals is influenced by crystal packing interactions. The presence of the Nδ1His-NGln hydrogen bond along with pH dependences of the peptide affinities suggest that deprotonation of the peptide His is required for high affinity binding of HPQ-containing peptides to streptavidin both in the crystals and in solution.

Screening of peptide libraries either displayed on phage by molecular biology or produced by combinatorial chemical synthesis is an effective method for discovery of peptide ligands for diverse protein targets. Owing to the remarkably high stability and high affinity of streptavidin for its natural ligand biotin (K d ϳ10 Ϫ15 M) (1), together with widespread bioanalytical, diagnostic, and therapeutic applications (2)(3)(4)(5), this protein has been extensively used to develop and validate such screening methodologies. High affinity unnatural ligands have been discovered by screening linear (6 -11) and cyclic (6) peptide libraries. Streptavidin also provides an ideal paradigm for probing the structural basis of high affinity protein-ligand interactions (12)(13)(14)(15)(16) and for introducing or improving properties by protein engineering (2,17,18). Finally, the high resolution crystal structures of apostreptavidin and of streptavidin-ligand complexes provide a powerful basis for developing structure-based ligand design strategies (12, 19 -23).
Recently we described the structures of streptavidin-bound linear and cyclic peptide ligands containing the HPQ sequence (13) discovered by phage display and probed the structural basis for the higher affinities of the cyclic ligands compared with the corresponding linear ones. These structures enabled the successful design of cyclic peptide ligands conformationally constrained with designed thioether cross-links (22), of streptavidin dimerizing peptide ligands (19 -21), and of a streptavidinbinding small molecule ligand (12).
The binding to streptavidin and avidin of certain small molecule and peptide ligands is pH-dependent. The affinities for avidin of biotin derivatives, 2-iminobiotin and diaminobiotin, decrease dramatically as the pH is lowered (24), as does the affinity for streptavidin of a linear peptide discovered by phage display, FSHPQNT (25). The extent of topochemical catalysis of disulfide formation and the resulting dimerization of designed streptavidin-bound HPQ-containing ligands whose thiols are presented next to one another in the crystal lattice also depend on pH (20).
Determination of the pH dependence of ligand binding or of changes in properties incurred by ligand binding often yields insight into the mode of action (26 -28) or mechanism of binding (29 -36) in biological and chemical processes. Appraisal of the ionization states of groups in a protein-bound ligand or at the ligand binding site of the protein target may reveal some of the determinants of high affinity binding crucial for structurebased ligand design. To this end we determine the pH dependences of binding to streptavidin of linear and cyclic peptide ligands containing the HPQ sequence and probe the structural basis for the dependences through crystallographic determination of complexes at multiple pH values. The affinities for streptavidin at pH 3.0, 4.0, 5.0, 6.0, 6.2, 6.4, 7.0, 7.3, 8.0, and 9.3 of linear Ac-AEFSHPQNTIEGRK-NH 2 , cyclo-Ac-AE[CH-PQGPPC]IEGRK-NH 2 , cyclo-Ac-AE[CHPQFC]IEGRK-NH 2 , and cyclo-[5-S-valeramide-HPQGPPC]K-NH 2 are determined by plasmon resonance measurements. We also determine the streptavidin-bound crystal structures of cyclo-[5-S-valeramide-HPQGPPC]K-NH 2 at pH 1.5, 2.5, 3.0, and 3.5 and of smaller versions of the other ligands, cyclo-Ac-[CHPQFC]-NH 2 at pH 2.0, 3.0, 3.6, 4.2, 4.8, and 11.8, cyclo-Ac-[CHPQGPPC]-NH 2 at pH 2.5, 2.9 and 3.7, and FSHPQNT at pH 4.0. Several structures are determined in two space groups, I222 and I4 1 22. The structures of the complexes are compared with those previously determined at other pH values. The crystal structures of the streptavidin-bound linear and cyclic peptide ligands determined over a range of pH values show that the pK a of the peptide His is greatly reduced in the crystals. Together with the pH dependences of the peptide affinities, the structures of the complexes over a range of pH values suggest that deprotonation of the peptide His is required for high affinity binding both in the crystals and in solution.
Crystallization of Streptavidin-Peptide Complexes-Apostreptavidin, purchased from Calbiochem, was crystallized by vapor diffusion in 40 sitting drops under conditions described for crystals of space group I222 (13,37). Streptavidin-FSHPQNT was cocrystallized (13)  The target pH values were obtained by adjusting 2-ml volumes of (NH 4 ) 2 SO 4 /buffer solution with NaOH or HCl using a calibrated Corning pH meter. The peptides were subsequently dissolved in smaller (ϳ100 -250 ) volumes whose final target pH values were confirmed with pH paper designed to measure integral pH values as well as pH values 0.5 units from integral. Because of the relatively large molarity of the buffers in the synthetic mother liquors and the smaller molarity of peptides, the effect of dissolving the peptide in the (NH 4 ) 2 SO 4 /buffer solutions is insignificant.
Crystallographic Data Collection and Refinement of Streptavidin-Ligand Complexes-Some x-ray diffraction data sets of streptavidinpeptide complexes were collected on a Siemens IPC area detector coupled to a Siemens three-circle goniometer mounted on a Rigaku rotating anode target copper tube operating at 50 kV, 60 mA. Data were indexed and reduced to produce integrated intensities and structure factors with the programs Sadie and Saint supplied by Siemens as described (13).
Other x-ray diffraction data sets were obtained with an R-AXIS IV image plate system equipped with mirrors mounted on the Rigaku generator upgraded to operate at 50 kV, 100 mA. The nonmonochromated mirror-focused x-rays were filtered with 0.004 cm of nickel. The image plate and associated data reduction software (Biotex) were from Molecular Structure Corporation (The Woodlands, TX). Crystal-to-detector distances ranged from 69.6 to 85.0 mm, 2 ϭ 0.0°, oscillations in (⌬) from 1.00 to 1.50°/frame, 20 -45 min/frame. For highly diffracting crystals, data were also collected at a distance of 75.7 mm, 2 ϭ 7.77°. Data collection statistics are summarized in Table I.
The previously determined crystal structures of streptavidin-peptide complexes (13,22) provided the starting structures for refinement of the complexes at other pH values. Structures were refined with X-PLOR (38) and with difference Fourier methods (39). In (͉F o ͉ Ϫ ͉F c ͉) ␣ c maps, positive and negative peaks whose magnitudes were greater than 2.8 were systematically identified with the program Peak-pick, which was written in house, and analyzed. Water structure was determined with Peak-pick, X-sight, or X-solvate from Molecular Simulations, Inc. (San Diego, CA) and refined according to published procedures (40). Waters with temperature factors greater than 50 Å 2 were examined in the context of the corresponding 2(͉F o ͉ Ϫ ͉F c ͉) ␣ c map and kept only if there was significant density for them. The coordinates corresponding to the conformations of discretely disordered residues (such as the peptide histidine in some structures) were simultaneously refined along with the rest of the structure, followed by simultaneous refinement of temperature factors and then of occupancies. Refinement of coordinates, temperature factors, and occupancies was iterated until the parameters and R-factor converged. Refinement statistics are given in Table I. The structures have been deposited into the Brookhaven Data Bank.
Determination of Affinities as a Function of pH by BIAcore Measurements-The affinities of linear and cyclic streptavidin binding peptides were determined as a function of pH by surface plasmon resonance. The Direct K d values were determined for cyclo-[5-S-valeramide-HPQG-PPC]K-NH 2 immobilized on the BIAcore chip. At each pH, two flow cell surfaces with different relative amounts of immobilized peptide were made by varying the peptide injection time. The cell with the lower density surface was used as a blank. At each pH, triplicate data sets were analyzed by the method of multispot sensing (41), which relies on equilibrium affinity measurements on two surfaces of different peptide ligand densities. To minimize perturbations to the apparent affinity constants caused by avidity or ligand binding to the surface more than once, it was necessary to use surfaces whose peptide densities corresponded to below 15 resonance units. Affinities within experimental error of those previously determined by other methods (6) were thus obtained.

Affinities of Core Peptides Used for Crystallography Are Similar to Affinities of Corresponding Longer Peptides Used for
BIAcore-Because of the large amounts of peptides required for both crystallography and BIAcore at low pH where the binding is weak, two sets of peptides were used for most of this study: core peptides for crystallography and longer peptides synthesized for a previous study (6) for BIAcore. The core peptides were used for crystallography because the C-and N-terminal extensions in the streptavidin-bound longer versions are expected to be disordered or to make crystal cracking from peptide soaking more likely. The affinities of the longer peptides were determined to be within experimental error of the corresponding core versions; the K d of cyclo-Ac-[CHPQGPPC]-NH 2 , pH 7.3, is 310 nM compared with 230 nM for cyclo-Ac-AE[CH-PQGPPC]IEGRK-NH 2 , pH 7.3 (22), and the K d of FSHPQNT is 78 mM (6) or 125 mM (25) at pH 7.3 compared with 150 mM for FSHPQNTK-NH 2 , pH 7.3 (12) or 160 mM determined here for Ac-AEFSHPQNTIEGRK-NH 2 , pH 7.3. Similarly the K d values of the other core peptides in this study were determined to lie within experimental error of the corresponding longer versions. An engineered thioether cross-linked ligand (22), cyclo-[5-Svaleramide-HPQGPPC]K-NH 2 , was used both for crystallography and BIAcore. pH-dependent Affinities Implicate Ionization of a Group with a pK a of ϳ6.3 upon Binding-The pH dependences of affinities of Ac-AEFSHPQNTIEGRK-NH 2 , cyclo-Ac-AE[CHPQGP-PC]IEGRK-NH 2 , cyclo-Ac-AE[CHPQFC]IEGRK-NH 2 , and cyclo-[5-S-valeramide-HPQGPPC]K-NH 2 determined by plasmon resonance measurements are shown in Table II and plotted in Fig. 1. The affinity of the linear peptide is several hundred-fold lower than the affinities of the cyclic peptides at all pH values of this study. The K d values of the ligands increase at roughly the same rate as the pH is lowered from ϳ6.3 to ϳ4.5, by an average factor of 3.0 Ϯ 0.8 per decrease in pH unit. Below pH ϳ4.5 the increase in K d values becomes smaller, and above pH ϳ6.3 the K d values become relatively pH-independent. The apparent pK a of ϳ6.3 is most clearly seen in the data for cyclo-[5-S-valeramide-HPQGPPC]K-NH 2 , for which direct K d values (and associated standard deviations from triplicate data sets) at pH 5.0, 6.0, 6.2, 6.4, 7.0, 7.3, 8.0, and 9.0 were determined. Thus ionization of a group (or groups) with an apparent pK a of ϳ6.3 in the unbound state is implicated in the binding process.
An Intrapeptide Hydrogen Bond between N␦1 His and N Gln Is Preserved at Low pH-The (2͉F o ͉ Ϫ ͉F c ͉) ␣ c map superimposed on the refined structures of I222 streptavidin-cyclo-[5-S-valeramide-CHPQGPPC]K-NH 2 , pH 2.5, at one of the two crystallographically independent ligand binding sites is shown in Fig.  2A. The refined N␦1 His -N Gln distance and associated angles (Table III) indicate a hydrogen bond between N Gln and the unprotonated N␦1 atom of the uncharged His. The imidazole density is strong, well defined, and flat, typical for a well ordered histidine. Similar density and an N␦1 His -N Gln distance and associated bond angles indicating a hydrogen bond are also obtained for the I4 1 22 complex at pH 2.5 and for the complexes of other linear and cyclic HPQ-containing peptides at pH values Ͼ 2.5 (Table III).
Crystal Structures at pH Values Յ 2.0 Show Protonated Histidine Components in Streptavidin-bound Peptides-The density for the peptide imidazole in streptavidin-cyclic peptide complexes at pH values Յ 2.0 indicates disorder. In I4 1 22 streptavidin-cyclo-Ac-[CHPQFC]-NH 2 , pH 2.0, the imidazole density becomes somewhat cylindrical through disorder involving an additional protonated conformer that is rotated by ϳ20°a bout the peptide histidine C␤-C␥ bond compared with the single unprotonated conformation in the same complex at pH 3.0 or in I222 streptavidin-cyclo-[5-S-valeramide-HPQGP-PC]K-NH 2 , pH 2.5 ( Fig. 2A). For the charged conformer the N␦1 His -N Gln hydrogen bond is no longer possible, and the N␦1 His -N Gln distance increases to 3.25 Å. The occupancy of the charged conformer in I4 1 22 cyclo-Ac-[CHPQFC]-NH 2 , pH 2.0, is 26%, yielding a calculated pK a of 1.5. Accompanying the disorder in the peptide imidazole in this complex is an increase in its average temperature factor from 28.4 Ϯ 0.5 Å 2 at pH 3.0 to 61.7 Ϯ 1.4 Å 2 at pH 2.0.
Disorder and mobility in the peptide His are also apparent at both ligand binding sites in I222 streptavidin-cyclo-[5-S-valeramide-HGPQFC]K-NH 2 , pH 1.5, in which the temperature factor of the peptide imidazole is high, ϳ63 Å 2 . The density for the peptide is weak and/or broken, reflecting weaker binding at  a Restrained, isotropic temperature factors were refined, and bulk solvent contributions were included for all structures. For I4 1 22 and I222 structures with more than 2000 and 4000 atoms, respectively, hydrogens were included in the refinements. For other structures polar hydrogens were included in the force field during refinement but were not included in the structure factor or map calculations and are not included in the total atom counts of these structures.
b Not including waters. When two values are given the first refers to disordered residues within the loop comprising residues 60 -69. Some or all of these residues were simultaneously refined in two conformations for the unique subunit of the I4 1 22 structures or for each of the two crystallographically independent subunits of the I222 structures. The disorder in the loops is incurred when Asp 61 , involved in an intersubunit hydrogen bonded salt bridge with His 87 at neutral pH, becomes protonated at low pH.
c Also includes ligand groups. Density for all side chain atoms or for terminal atoms in these groups was weak or absent, and temperature factors were high. Occupancies (occs) for poorly defined groups of atoms were refined. Discretely disordered groups are not included in this category.
d Siemens (X-1000) data with R sym Ͼ 50% were rejected along with data with values Ͼ3.5 from the mean for each set of symmetry equivalents. R-AXIS IV data were rejected if ( for reflections from 7.5 Å to the highest resolution). g Cross-validation R-factor using 10% of the data withheld from the refinement (62). h Root mean square deviations from ideal bond lengths and bond angles and torsions. i Head-to-tail disulfide bonded peptide dimer (19).
this low pH, but the density of the imidazole is well defined enough to resolve two imidazole components. The N␦1 atom of one component is not within hydrogen bonding distance of N Gln and is surely protonated. Although the refined N␦1 His -N Gln distance (2.92 Ϯ 0.13 Å) of the other component suggests a hydrogen bond, the N Gln -H Gln -N␦1 His angle is poor (123 Ϯ 5°), lower by 4.1 than the average for the other complexes above pH 2.0 (Table III). Thus this other component may be largely protonated as well, despite the small N␦1 His -N Gln distance. Ionization State of the Peptide Histidine Depends on Crystal Packing-In I222 streptavidin-peptide complexes one of the two crystallographically independent binding sites (site 1) is near a 2-fold related crystallographically equivalent site and is thus more shielded from solvent than site 2. The unique site in I4 1 22 streptavidin is also solvent-shielded from a nearby 2-fold related equivalent site. At many pH values the density of the bound peptide in the complexes is better defined at the unique site in I4 1 22 complexes and at site 1 in I222 complexes than at site 2. The conclusions regarding the protonation states of the His of the bound peptides are essentially the same for site 1 and site 2 of I222 complexes and for the unique site of I4 1 22 complexes at pH values Ͼ 2.5 where the density is well defined, the temperature factors relatively low, and the N␦1 His -N Gln distances and associated angles (Table III) clearly indicate an N␦1 His -N Gln hydrogen bond at all three sites. However, at pH values Յ pH 2.5, differences in the protonation state of the peptide His are observed at crystallographically different sites. Fig. 2 (A and B) compares the structures of the bound peptide at site 1 and site 2, respectively, for I222 streptavidin-cyclo-[5-S-valeramide-HPQGPPC]K-NH 2 , pH 2.5. The imidazole density at site 2 is distinctly different from that at site 1; it is elongated in a direction corresponding to the presence of a second, protonated histidine conformer that is rotated 17°a bout the C␣-C␤ bond with respect to the unprotonated conformer. The distance and angle parameters of the unprotonated conformer indicate an N␦1 His -N Gln hydrogen bond (Table III) shown in yellow in Fig. 2B. The occupancies of the protonated and unprotonated conformers are 20 and 80%, respectively, yielding a calculated pK a at site 2 of 1.9. For the same I222 complex at pH 3.0, similar inequivalence of the two sites is observed, with resolvable protonated and unprotonated conformers at site 2. The occupancies of the protonated and unprotonated conformers at site 2 are 15 and 85%, respectively, at pH 3.0, yielding a calculated pK a of 2.2. By contrast, at site 1, the occupancies of any unprotonated components at pH Ն 2.5 are not high enough to observe or to resolve. Thus the pK a
In the conformation of the protonated component at site 2 in I222 streptavidin-cyclo-[5-S-valeramide-HPQGPPC]K-NH 2 , pH 2.5, shown in Fig. 2B, the side chain is rotated 180°about the C␤-C␥ bond with respect to the conformation of the unprotonated component to allow N␦1 to make a hydrogen bond (shown in cyan) with a water molecule. This change in the His conformation increases the N⑀2 His -O␥ Ser88 distance from 3.05 to 3.21 Å and decreases the N⑀2 His -H⑀2 His -O␥ Ser88 angle from 163 to 108°, indicating a weakening or loss of the hydrogen bond involving N⑀2 His and O␥ Ser88 . However an alternate hydrogen bond, C⑀1 His -O␥ Ser88 ϭ 2.98 Å, C⑀1 His -H⑀1 His -O␥ Ser88 ϭ 126°(shown in cyan in Fig. 2B) similar to those observed in other protein crystal structures (42) is now possible.
Inequivalence of the two peptide binding sites in I222 complexes is also manifested by differences in temperature factors for the bound ligands at many pH values. Temperature factors are lower at site 1 than at site 2 except at low pH where they become large at both sites. For example, in the I222 cyclo-[5-S-valeramide-HPQGPPC]K-NH 2 complex the average temperature factor of the imidazole at pH 2.5, 3.0, and 3.5 is lower at site 1 (21 Ϯ 4 Å 2 ) than at site 2 (36 Ϯ 5 Å 2 ), whereas at pH 1.5 it increases dramatically at both sites, becoming the same at site 1 (63 Ϯ 2 Å 2 ) as at site 2 (64 Ϯ 2 Å 2 ). For the I4 1 22 complexes, the ligand temperature factors are as low as at site 1 in the I222 complexes.

Binding to Streptavidin of HPQ-containing Peptide Ligands
Involves Deprotonation of the Peptide Histidine-The pH dependences of affinities for streptavidin of linear and cyclic HPQ-containing peptides are consistent with ionization of a group with a pK a of ϳ6.3 in the unbound state involved in binding. The unperturbed pK a for the His side chain fully exposed to water is 6.3 (43). The observation of the N␦1 His -N Gln hydrogen bond over a range of pH values in the bound linear and cyclic peptides shows that the peptide His is uncharged and unprotonated at pH values as low as 2.5 in the crystalline cyclic peptide complexes; the pK a of the peptide His is thus reduced to a value of less than 2.5. Therefore the decrease in binding affinity in solution as the pH decreases is attributed to the cost of deprotonating the peptide His at low pH. The deprotonation and formation of the N␦1 His -N Gln hydrogen bond are required for high affinity binding both in the crystals and in solution.
Table III provides the N␦1 His -N Gln hydrogen bond lengths and associated angles for various HPQ-containing peptide ligands at various pH values for this and other investigations (13, 19 -22). This hydrogen bond is also observed in complexes of streptavidin with HHPQGPPH, linear (reduced) Ac-CHPQG-PPC-NH 2 , and linear Ac-CHPQFC-NH 2 . 2 These data suggest that for other HPQ-containing linear peptide ligands such as (HDHPQNL and SHPQGPPS) and disulfide-bonded cyclic peptide ligands such as cyclo-[CHPQFSNC], cyclo-[CHPQFPC], and cyclo-[CHPQFNC] (6), the same hydrogen bond forms in the complexes with streptavidin at pH values above 2.5.
Long Range Crystal Packing Interactions Perturb the pK a of the Histidine of the Bound Peptide Ligands-Inequivalence of the two crystallographically independent sites in I222 streptavidin results in significant differences in some of their properties. The temperature factors of bound peptide ligands are often lower and the density better defined at site 1 than at the more solvent exposed site 2. Topochemical lattice-mediated disulfide interchange occurs between neighboring bound cyclo-Ac-[CHPQGPPC]-NH 2 ligands at site 1, which is close to a 2-fold related equivalent site, but not at site 2 (21). Two of the hydrogen bonds to the ureido oxygen of biotin are systematically shorter over a range of pH values at site 1 than at site 2.  this investigation site 1 and site 2 were also shown to differ with respect to the protonation state of the His in bound HPQcontaining peptide ligands. The pK a of the peptide His is lower at the more solvent-shielded site 1 than at site 2. In the I4 1 22 space group the pK a at the unique, solvent-shielded ligand binding site is also lower than at the solvent exposed site 2 in the I222 space group. The protonation state of the peptide His at the more solvent exposed site 2 in I222 streptavidin-peptide crystals more accurately reflects its state in solution. However, because of the observed effect of crystal packing on the pK a of the peptide His at site 1 in I222 streptavidin complexes and at the unique site in I4 1 22 streptavidin complexes, solvent shielding to a lesser extent by crystal packing at site 2 in I222 streptavidin complexes must also be considered as a potential factor that could perturb the pK a of the peptide His at this site from the pK a in solution. Thus the crystallographically determined pK a of the peptide His at site 2 in I222 streptavidin should be taken as a lower limit to the corresponding value in solution.
Directionalities of Hydrogen Bonds Involving Linear and Cyclic Peptide Ligands Are Unambiguous-Because the proton atoms of protein-ligand complexes are normally not visible by x-ray crystallography, directionalities of hydrogen bonding interactions can not always unambiguously be determined by this technique. In some cases, however, from the environments of residues or groups participating in hydrogen bond networks, directionalities of some hydrogen bonds can be inferred (44). For the complexes of streptavidin with the linear and cyclic peptide ligands discussed here, the directionality of every hydrogen bond involving every atom of each ligand is uniquely determined at each pH value Ͼ 2.5.
In complexes of streptavidin with HPQ-containing peptide ligands, the orientation of the peptide Gln side chain amide group is unambiguous based in part on the better geometry of hydrogen bonds in one orientation (Fig. 3A) versus the alternate one in which this amide is rotated by 180° (Fig. 3B). In the less favorable orientation (Fig. 3B), the vector between N⑀2 Gln and O␥2 Thr90 is directed right between the Gln N⑀2 hydrogens, and there are two proton donors to O␥2 Thr90 (N⑀2 Gln and N⑀1 Trp79 ). In the more favorable orientation, the O␥2 proton of Thr90 is directed at O⑀1 Gln , whereas one of the N⑀2 Gln hydro-gens is directed at the oxygen of Wat 600 . In this arrangement O␥2 Thr90 receives a proton from N⑀1 Trp79 and donates a proton to O⑀1 Gln . The angles associated with these hydrogen bonds are more favorable in this orientation than in the alternate one. The angle between the hydrogen bonds received and provided by O␥2 Thr90 is ϳ90°. The peptide Gln side chain is also uniquely oriented because of its interaction (d ϭ 3.51 Ϯ 0.15 Å, determined from 12 structures) with the Trp 108 ring, which reflects an NH 3 aromatic ring system hydrogen bond similar to those described (45).
FSHPQNT- Fig. 4 shows the hydrogen bonding network that connects the linear peptide to streptavidin at pH 4.0 and 5.6. The orientation of the peptide Asn side chain is unambiguous because of the hydrogen bond between its O␦1 atom and N␦2 Asn23 . Asn 23 is in turn uniquely oriented due to the hydrogen bond between its O␦1 atom and N Ser27 . The peptide Asn side chain is also uniquely oriented because of an N␦2 Asn23 3 Trp120 interaction (d ϭ 3.64 Ϯ 0.04 Å, determined from four structures). Because the peptide His N␦1 atom accepts a proton from the peptide Gln main chain NH group, the peptide His N⑀2 atom must donate a proton to O␥ Ser88 . The two other peptide groups, O Gln and N Thr , involved in hydrogen bonds must be an acceptor and donor, respectively. Thus the directionalities of all 11 peptide-protein hydrogen bonds in streptavidin-FSHPQNT are unambiguous, as well as the directionality of the intrapeptide hydrogen bond.
In a previous study of the binding of streptavidin to FSH-PQNT, it was suggested that enhanced binding at neutral compared with acidic pH reflects an equilibrium between alternate unprotonated states of the peptide His in alternate equi-energetic hydrogen bond networks involving the protein at neutral pH (25). Thus an increase in binding at neutral pH was proposed to reflect an increase in entropy (25). In the present study, however, we were able to establish one unique hydrogen bonding network wherein the directionalities of all hydrogen bonds between FSHPQNT and streptavidin, even those mediated by water molecules, are essentially unambiguous at pH 4.0 and 5.6. Similarly, unique, lowest energy hydrogen bond networks were delineated for all the streptavidincyclic peptide complexes at pH values Ͼ 2.5. In this investigation any disorder in the His occurs at low pH (Ͻ 2.5), not neutral or basic pH. Therefore the pH dependence of binding of any of these peptides to streptavidin is probably not due to such an entropy effect. The structures and affinities of the linear and cyclic HPQ-containing peptide ligands determined over a large range of pH values in two space groups are most consistent with deprotonation of the peptide His upon binding as a major determinant of the pH dependence of ligand binding. Structural Basis for the Large Perturbation of the pK a of the Peptide Histidine-The experimentally determined pK a values of certain noncatalytic residues in naturally occurring proteins are shifted by as much as 2.5 units (46). Larger shifts in the pK a values often occur in functionally important residues, such as those at active sites where buried charged residues often participate in catalysis (47)(48)(49)(50)(51). For example, the pK a of active site Asp 26 in reduced thioredoxin is elevated by more than 5 units (52). The pK a values of charged residues engineered into hydrophobic cores of protein mutants are also perturbed by as much as 3.9 units (53)(54)(55). Much of the theoretical work that involves prediction of pH-dependent properties of proteins is based on the assumption that ionization equilibria in proteins are influenced primarily by electrostatic interactions (43, 56 -58). Hydrogen bonding interactions involving nonionizable groups (59) and hydrophobic interactions (60) may also play roles in determining pK a values. Large shifts in pK a values can also be effected simply by desolvation, the dominant factor shifting the pK a of the buried lysine introduced into staphylococcal nuclease (53).
Although the peptide His side chain of streptavidin-bound HPQ-containing peptide ligands is not involved in salt bridge interactions, it makes two hydrogen bonds (N␦1 His -N Gln and N⑀2 His -O␥2 Thr90 ) at pH values Ն 2.5. Thus, hydrogen bonding interactions together with desolvation within a protein cavity of low dielectric constant are among the factors perturbing the pK a in the streptavidin-HPQ-containing peptide complexes. Although the C␦2 atom of the imidazole of the peptide His is solvent-accessible from one direction, the other imidazole atoms are shielded from solvent by the rest of the bound peptide and by Trp 79 , Leu 110 , Ser 88 , Ala 86 , and Trp 120 of a neighboring subunit. Upon protonation at low pH a small rotation about 1 of the peptide His and a 180°rotation about 2 allows N␦1 to hydrogen bond with a solvent molecule (Fig. 2B).
Conclusions-Through plasmon resonance measurements combined with crystallography at multiple pH values on a set of HPQ-containing ligands, features of the mechanism of high affinity binding to streptavidin have been delineated. High resolution crystal structures at pH values as low as 1.5 yield insight into the nature of the structural rearrangements that occur in the bound peptide upon protonation of the His at three crystallographically different binding sites in two space groups. Observation of perturbations to the pK a of the peptide His from long range crystal packing interactions should be taken as a caveat in extrapolation of pK a values determined in crystals to the corresponding ones in solution. The determination of the greatly reduced pK a of the His in streptavidin-bound HPQcontaining peptides and of the difference in pK a at sites with different extents of solvent shielding should provide valuable structural data for testing and improving theoretical models directed at predicting pH-dependent properties of proteins and of protein-ligand complexes.