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

(Received for publication, January 28, 1997, and in revised form, March 6, 1997)

Bradley A. Katz Dagger and Robert T. Cass

From Arris Pharmaceutical Corporation, South San Francisco, California 94080

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES


ABSTRACT

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, the Kd 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. The Kd 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 in Kd values, and above pH ~6.3 the Kd 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 Ndelta 1 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 pKa of the bound peptide His in the crystals is influenced by crystal packing interactions. The presence of the Ndelta 1His-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.


INTRODUCTION

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 (Kd ~10-15 M) (1), together with widespread bioanalytical, diagnostic, and therapeutic applications (2-5), 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 (12-16) 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 streptavidin-binding 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 structure-based 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-NH2, cyclo-Ac-AE[CHPQGPPC]IEGRK-NH2, cyclo-Ac-AE[CHPQFC]IEGRK-NH2, and cyclo-[5-S-valeramide-HPQGPPC]K-NH2 are determined by plasmon resonance measurements. We also determine the streptavidin-bound crystal structures of cyclo-[5-S-valeramide-HPQGPPC]K-NH2 at pH 1.5, 2.5, 3.0, and 3.5 and of smaller versions of the other ligands, cyclo-Ac-[CHPQFC]-NH2 at pH 2.0, 3.0, 3.6, 4.2, 4.8, and 11.8, cyclo-Ac-[CHPQGPPC]-NH2 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 I4122. 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 pKa 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.


EXPERIMENTAL PROCEDURES

Peptide Synthesis

Peptides for crystallography, FSHPQNT, cyclo-Ac-[CHPQGPPC]-NH2, cyclo-Ac-[CHPQFC]-NH2, and cyclo-[5-S-valeramide-HPQGPPC]K-NH2 were synthesized as described (6, 13, 22). For BIAcore analysis, corresponding peptides Ac-AEFSHPQNTIEGRK-NH2, cyclo-Ac-AE[CHPQGPPC]IEGRK-NH2, cyclo-Ac-AE[CHPQFC]IEGRK-NH2 and cyclo-[5-S-valeramide-HPQGPPC]K-NH2 were synthesized as described (6, 22). The first three of these are longer than the corresponding ones for crystallography; they contains N-terminal AE sequence and C-terminal IEGRK for reasons independent of the present study (6).

Crystallization of Streptavidin-Peptide Complexes

Apostreptavidin, purchased from Calbiochem, was crystallized by vapor diffusion in 40 lambda  sitting drops under conditions described for crystals of space group I222 (13, 37). Streptavidin-FSHPQNT was cocrystallized (13) in the I222 and I4122 (a = 58.0 Å, c = 174.5 Å) space groups under similar conditions, pH 4.0, at an initial peptide concentration of 15.0 mg/ml. Streptavidin-cyclo-Ac-[CHPQFC]-NH2 crystals, pH 2.0, 3.0, 3.6, 4.2, 4.8, and 11.8 were prepared by soaking I4122 streptavidin-cyclo-Ac-[CHPQFC]-NH2 cocrystals (13) in synthetic mother liquor (75% saturated (NH4)2SO4, 25% buffer) at peptide concentrations ranging from 15.0 to 28.0 mg/ml for several days. For the pH 2.0 and 3.0 structures the buffer was 1.0 M sodium formate. At pH 3.6, 4.2, and 4.8 the buffer was 1.0 M potassium acetate, and at pH 11.8 it was 0.10 M CAPS.1 Crystals of streptavidin-cyclo-[5-S-valeramide-HPQGPPC]K-NH2, and -cyclo-Ac-[CHPQGPPC]-NH2 were prepared by soaking I222 or I4122 apostreptavidin crystals in 75% saturated (NH4)2SO4, 0.25 M sodium formate, 20 mg/ml peptide, pH 1.5, 2.5, or 2.9, or in 75% (NH4)2SO4, 0.25 M potassium acetate, pH 3.7.

The target pH values were obtained by adjusting 2-ml volumes of (NH4)2SO4/buffer solution with NaOH or HCl using a calibrated Corning pH meter. The peptides were subsequently dissolved in smaller (~100-250 lambda ) 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 (NH4)2SO4/buffer solutions is insignificant.

Crystallographic Data Collection and Refinement of Streptavidin-Ligand Complexes

Some x-ray diffraction data sets of streptavidin-peptide 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, 2theta  = 0.0 °, oscillations in phi  (Delta phi ) 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, 2theta  = 7.77 °. Data collection statistics are summarized in Table I.

Table I. Crystallography of streptavidin-peptide complexes at various pH values


Parameters, cyclo-Ac-[CHPQFC]-NH2a
  Area detector system X-1000 X-1000 R-AXIS IV R-AXIS R-AXIS X-1000
  pH 2.00 3.00 3.60 4.20 4.77 11.80
  Space group I4122 I4122 I4122 I4122 I4122 I4122
  No. atoms (including disorder) 1052 1053 2153 2119 2248 1074
  No. waters (including disorder) 65 62 45 34 77 53
  No. discretely disordered groupsb 4 6 10 + 4b 10 + 4 10 + 4 8
  No. residues with refined occsc 23 24 24 25 26 23
Diffraction statistics
  Resolution (Å) 1.79 1.81 1.33 1.33 1.32 1.72
  No. observationsd 49532 49331 126387 85713 61076 49797
  No. merged reflections 12424 12200 26406 28233 20636 13754
  Average redundancy 3.9 4.0 4.8 3.0 3.0 3.6
  Rmerge (%)e 5.3 6.0 8.1 9.4 9.3 7.5
Refinement statistics
  Refinement resolution 7.5-1.86 7.5-1.87 7.5-1.50 7.5-1.60 7.5-1.85 7.5-1.82
  No. merged reflections 10046 10462 18080 13205 10404 10850
  |Fo|/sigma cut-off 2.0 2.0 2.7 1.7 2.6 2.0
  Rcryst (%)f 19.1 17.9 20.3 19.8 20.5 18.3
  Free Rcryst (%)g 20.9 20.9 24.6 24.3 25.0 23.9
  Overall completeness (%) 75.0 79.3 74.2 65.3 80.5 75.9
  At highest resolution (%) 28.9 36.4 32.6 38.5 60.5 38.4
  Highest resolution shell 1.94-1.86 1.95-1.87 1.57-1.50 1.67-1.60 1.93-1.85 1.90-1.82
Root mean square deviationsh
  Bond lengths (Å) 0.019 0.018 0.018 0.017 0.019 0.017
  Bond angles (°) 3.0 3.0 4.2 4.3 4.4 2.8
  Torsion angles (°) 28.5 28.4 25.8 25.3 25.1 27.6
cyclo-Ac-[CHPQGPPC]-NH2 monomer monomer monomer dimeri dimeri FSHPQNT
  Area detector system R-AXIS IV R-AXIS X-1000 R-AXIS R-AXIS X-1000
  pH 2.50 2.85 3.67 2.50 3.50 4.00
  Space group I4122 I4122 I4122 I4122 I4122 I222
  No. atoms (including disorder) 1049 2242 1143 2107 2248 2134
  No. waters 60 54 76 56 58 179
  No. discretely disordered groupsb 4 10 + 7 10 + 7 6 10 + 7 4
  No. residues with refined occsc 22 25 22 31 24 27
Diffraction statistics
  Resolution (Å) 1.35 1.38 1.84 1.33 1.33 1.63
  No. observationd 34337 103204 38381 65678 45610 98073
  No. merged reflections 17544 27131 12118 25029 26527 25792
  Average redundancy 2.0 3.8 3.2 2.6 1.7 3.8
  Rmerge (%)e 8.0 6.0 8.0 6.0 5.9 8.3
Refinement statistics
  Refinement resolution 7.5-1.75 7.5-1.65 7.5-1.92 7.5-1.46 7.5-1.48 7.5-1.85
  No. merged reflections 10179 12808 9723 18850 14708 15514
  |Fo|/sigma cut-off 2.0 2.4 2.0 1.9 1.9 2.0
  Rcryst (%)f 17.9 19.9 19.1 20.1 19.8 19.2
  Free Rcryst (%)g 23.5 22.8 23.6 23.7 23.9 25.9
  Overall completeness (%) 65.7 70.5 77.5 72.2 57.9 71.4
  At highest resolution (%) 40.8 34.6 36.9 40.0 27.9 42.0
  Highest resolution shell 1.83-1.75 1.72-1.65 1.95-1.92 1.53-1.46 1.55-1.48 1.93-1.85
Root mean square deviationsh
  Bond lengths (Å) 0.018 0.020 0.019 0.020 0.019 0.017
  Bond angles (°) 3.3 4.3 3.0 4.5 4.3 3.4
  Torsion angles (°) 28.5 26.1 27.4 26.4 26.2 27.7
Parameters, cyclo-[5-S-valeramide-HPQGPPC]K-NH2a
  Area detector system R-AXIS IV R-AXIS R-AXIS R-AXIS R-AXIS R-AXIS
  pH 1.50 2.50 2.50 3.00 3.50 3.50
  Space group I222 I222 I4122 I222 I4122 I222
  No. atoms (including disorder) 4352 4554 2223 4680 2266 4683
  No. waters 146 139 64 171 70 185
  No. discretely disordered groupsb 11 20 + 9 10 + 2 20 + 9 10 + 4 20 + 9 
  No. residues with refined occsc 46 30 24 30 24 30
Diffraction statistics
  Resolution (Å) 1.32 1.35 1.33 1.33 1.33 1.32
  No. observationsd 89532 97859 127962 82155 108525 79817
  No. merged reflections 41504 36758 28160 41763 27343 42836
  Average redundancy 2.2 2.7 4.5 2.0 4.0 1.9
  Rmerge (%)e 5.6 5.4 10.7 7.9 6.8 7.0
Refinement statistics
  Refinement resolution 7.5-1.50 7.5-1.45 7.5-1.70 7.5-1.45 7.5-1.50 7.5-1.45
  No. merged reflections 25381 29499 11983 28570 17621 27282
  |Fo|/sigma cut-off 1.8 1.8 2.5 1.6 2.0 1.7
  Rcryst (%)f 20.2 20.7 19.5 20.5 20.6 20.0
  Free Rcryst (%)g 23.5 23.7 22.3 24.0 24.6 24.4
  Overall completeness (%) 65.7 68.8 71.6 66.8 72.4 64.0
  At highest resolution (%) 34.3 43.3 48.4 33.6 39.7 32.5
  Highest resolution shell 1.57-1.50 1.52-1.45 1.78-1.70 1.52-1.45 1.57-1.50 1.52-1.45
Root mean square deviationsh
  Bond lengths (Å) 0.019 0.020 0.017 0.019 0.017 0.019
  Bond angles (°) 4.3 4.3 4.3 4.3 4.2 4.3
  Torsion angles (°) 24.9 25.1 25.3 25.3 25.4 25.3

a Restrained, isotropic temperature factors were refined, and bulk solvent contributions were included for all structures. For I4122 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 I4122 structures or for each of the two crystallographically independent subunits of the I222 structures. The disorder in the loops is incurred when Asp61, involved in an intersubunit hydrogen bonded salt bridge with His87 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 Rsym > 50% were rejected along with data with values >3.5 sigma  from the mean for each set of symmetry equivalents. R-AXIS IV data were rejected if (I(h)i - <I(h)i>) > [0.30* (< global I> ) + 0.10*I(h)i], where I(h)i is the ith observation of the intensity of reflection h (61).
e Rmerge = Sigma hSigma i|I(h)i - < I(h)> |/Sigma hSigma iI(h)i.
f Rcryst = Sigma  (||Fo- |Fc||)/Sigma |Fo| (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).

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 (|Fo- |Fc|) alpha c maps, positive and negative peaks whose magnitudes were greater than 2.8 sigma  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 Å2 were examined in the context of the corresponding 2(|Fo- |Fc|) alpha 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 BIAcore 2000 system, sensor chip, amine coupling kit containing N-hydroxysuccinimide, N-ethyl-N'-(3-diethylaminopropyl)-carbodiimide, and ethanolamine-HCl were from Pharmacia Biotech Inc. Buffers containing 150 mM NaCl, 3.4 mM EDTA, and 0.0050% surfactant P20 ranging from pH 3.0 to 8.0 were 10 mM potassium acetate, pH 3.0; 10 mM sodium acetate, pH 4.0, 5.0, and 6.0; 10 mM bis-Tris, pH 6.2 and 6.4; 10 mM HEPES, pH 7.0 and 7.3; and 10 mM Tris, pH 8.0 and 9.0.

The peptide, cyclo-Ac-AE[CHPQGPPC]IEGRK-NH2, was coupled directly to the sensor chip surface via the epsilon -amino group of the C-terminal lysine residue under a continuous HEPES buffer flow of 10 µl/min. The sensor chip surface was activated with a 2-min pulse of a solution of 50 mM N-hydroxysuccinimide, 200 mM N-ethyl-N'-(3-diethylaminopropyl)-carbodiimide. The peptide surface was made by injection of 100 mM peptide in 100 mM sodium borate, pH 8.5. To inactivate any remaining N-hydroxysuccinimide ester groups, the immobilization procedure was completed by an 8-min injection of 1.0 M ethanolamine-HCl followed by a 2-min pulse of 6.0 M guanidine-HCl, pH 2.1, to wash out any noncovalently bound peptide. The Kd values of cyclo-Ac-AE[CHPQGPPC]IEGRK-NH2, cyclo-AE[CHPQFC]IEGRK-NH2, and Ac-AEFSHPQNTIEGRK-NH2 were determined by described procedures (13, 20, 22) as a function of pH through competition for streptavidin with cyclo-Ac-AE[CHPQGPPC]IEGRK-NH2 immobilized on the surface.

Direct Kd values were determined for cyclo-[5-S-valeramide-HPQGPPC]K-NH2 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.


RESULTS

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 Kd of cyclo-Ac-[CHPQGPPC]-NH2, pH 7.3, is 310 nM compared with 230 nM for cyclo-Ac-AE[CHPQGPPC]IEGRK-NH2, pH 7.3 (22), and the Kd of FSHPQNT is 78 mM (6) or 125 mM (25) at pH 7.3 compared with 150 mM for FSHPQNTK-NH2, pH 7.3 (12) or 160 mM determined here for Ac-AEFSHPQNTIEGRK-NH2, pH 7.3. Similarly the Kd 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-S-valeramide-HPQGPPC]K-NH2, was used both for crystallography and BIAcore.

pH-dependent Affinities Implicate Ionization of a Group with a pKa of ~6.3 upon Binding

The pH dependences of affinities of Ac-AEFSHPQNTIEGRK-NH2, cyclo-Ac-AE[CHPQGPPC]IEGRK-NH2, cyclo-Ac-AE[CHPQFC]IEGRK-NH2, and cyclo-[5-S-valeramide-HPQGPPC]K-NH2 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 Kd values of the ligands increase at roughly the same rate as the pH is lowered from ~6.3 to ~4.5, by an average factor of 3.0 ± 0.8 per decrease in pH unit. Below pH ~4.5 the increase in Kd values becomes smaller, and above pH ~6.3 the Kd values become relatively pH-independent. The apparent pKa of ~6.3 is most clearly seen in the data for cyclo-[5-S-valeramide-HPQGPPC]K-NH2, for which direct Kd 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 pKa of ~6.3 in the unbound state is implicated in the binding process.

Table II. Dissociation constants (M) as a function of pH for streptavidin of linear and cyclic HPQ-containing peptides


pH Cyclo-Ac-AE[CHPQGPPC]IEGRK-NH2 Cyclo-Ac-AE[CHPQFC]IEGRK-NH2 Cyclo-[5-S-valeramide-HPQGPPC]K-NH2 Ac-AEFSHPQNTIEGRK-NH2

× 10-9 × 10-9 × 10-9 × 10-6
3.0 2300 2200 ND ND
4.0 2200 ND 2330 ND
5.0 1400 1500 3100 (350)b 2100
6.0  380  500 1730 (400) 840
6.2 NDa ND 1400 (460) ND
6.4 NDa ND  687 (239) ND
7.0 NDa   45  284 (60) 160
7.3  150   23  256 (25) 160
8.0  130   47  322 (50) 230
9.0 NDa ND  373 (87) ND

a ND, not determined.
b Standard deviations determined from three measurements are in parentheses.


Fig. 1. Dissociation constants as a function of pH for binding to streptavidin of Ac-AEFSHPQNTIEGRK-NH2, cyclo-Ac-AE[CHPQGPPC]IEGRK-NH2, cyclo-Ac-AE[CHPQFC]IEGRK-NH2, and cyclo-[5-S-valeramide-HPQGPPC]K-NH2.
[View Larger Version of this Image (24K GIF file)]

An Intrapeptide Hydrogen Bond between Ndelta 1His and NGln Is Preserved at Low pH

The (2|Fo- |Fc|) alpha c map superimposed on the refined structures of I222 streptavidin-cyclo-[5-S-valeramide-CHPQGPPC]K-NH2, pH 2.5, at one of the two crystallographically independent ligand binding sites is shown in Fig. 2A. The refined Ndelta 1His-NGln distance and associated angles (Table III) indicate a hydrogen bond between NGln and the unprotonated Ndelta 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 Ndelta 1His-NGln distance and associated bond angles indicating a hydrogen bond are also obtained for the I4122 complex at pH 2.5 and for the complexes of other linear and cyclic HPQ-containing peptides at pH values > 2.5 (Table III).


Fig. 2. (2|Fo- |Fc|) alpha c map for I222 streptavidin-cyclo-[5-S-valeramide-HPQGPPC]K-NH2, pH 2.5, site 1 (top) or site 2 (bottom) superimposed on the refined structure. At site 2 hydrogen bonds associated with the major conformer are yellow; those of the minor conformer are cyan. Note that Leu110 is discretely disordered between two low energy conformations.
[View Larger Version of this Image (172K GIF file)]

Table III. Hydrogen bond length (Ndelta 1His-NGln) and associated angles for streptavidin-bound HPQ-containing peptides at various pH values


ligand pH Space group Resolution Length Angle 1a Angle 2b reference

Å Å degrees degrees
Cyclo-Ac-[CHPQFC]-NH2 2.00 I4122 1.86 2.68c 133 139 this work
3.00 I4122 1.87 2.65 157 144 this work
3.60 I4122 1.50 2.76 149 158 this work
4.20 I4122 1.60 2.80 142 158 this work
4.77 I4122 1.85 2.85 145 159 this work
7.50 I4122 2.00 2.86 153 166 13
11.80 I4122 1.82 2.69 145 156 this work
Cyclo-Ac-[CHPQGPPC]-NH2 2.50 I4122 1.75 2.70 149 157 this work
2.85 I4122 1.65 2.74 155 149 this work
3.67 I4122 1.92 2.87 157 139 this work
5.00 I222 2.00 2.96 (1)d 158 (9) 161 (8) 13
Cyclo-Ac-[CHPQGPPC]-NH2 dimer 2.50 I4122 1.46 2.59 157 144 this work
3.50 I4122 1.92 2.80 157 161 this work
5.00 I222 1.90 2.90 (1) 153 (7) 160 (2) 21
7.50 I4122 1.92 2.96 150 164 19
Cyclo-[5-S-valeramide-HPQGPPC]K-NH2 1.50 I222 1.50 2.92 (13)c 169 (1) 123 (5) this work
2.50 I222 1.45 2.86 (8)c 160 (6) 149 (9) this work
2.50 I4122 1.50 2.81 154 153 this work
3.00 I222 1.50 2.89 (1)c 160 (5) 153 (13) this work
3.50 I4122 1.50 2.87 152 160 this work
3.50 I222 1.45 2.93 (4) 165 (8) 144 (10) this work
[4-S-toluamide-HPQGPPC]-NH2 6.00 I222 1.92 2.88 (4) 152 (0) 162 (4) 22
FSHPQNT 4.00 I222 1.85 2.88 (11) 149 (2) 157 (10) this work
5.60 I222 1.76 2.87 (2) 150 (2) 156 (4) 13
FCFPQNT-NH2 dimer 7.00 I222 1.95 3.07 (6) 154 (3) 162 (10) 20
Ac-CFPQNT-NH2 dimer 7.00 I222 1.80 3.03 (4) 152 (3) 160 (8) 20

a Imidazole centroidHis-Ndelta 1His-NGln.
b NGln-HGln-Ndelta 1His.
c Values refer to components of disordered His that are within hydrogen bonding distance of NGln.
d For I222 structures, averages and standard deviation refer to the two crystallographically independent ligands.

Crystal Structures at pH Values <=  2.0 Show Protonated Histidine Components in Streptavidin-bound Peptides

The density for the peptide imidazole in streptavidin-cyclic peptide complexes at pH values <=  2.0 indicates disorder. In I4122 streptavidin-cyclo-Ac-[CHPQFC]-NH2, pH 2.0, the imidazole density becomes somewhat cylindrical through disorder involving an additional protonated conformer that is rotated by ~20 ° about the peptide histidine Cbeta -Cgamma bond compared with the single unprotonated conformation in the same complex at pH 3.0 or in I222 streptavidin-cyclo-[5-S-valeramide-HPQGPPC]K-NH2, pH 2.5 (Fig. 2A). For the charged conformer the Ndelta 1His-NGln hydrogen bond is no longer possible, and the Ndelta 1His-NGln distance increases to 3.25 Å. The occupancy of the charged conformer in I4122 cyclo-Ac-[CHPQFC]-NH2, pH 2.0, is 26%, yielding a calculated pKa of 1.5. Accompanying the disorder in the peptide imidazole in this complex is an increase in its average temperature factor from 28.4 ± 0.5 Å2 at pH 3.0 to 61.7 ± 1.4 Å2 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-NH2, pH 1.5, in which the temperature factor of the peptide imidazole is high, ~63 Å2. The density for the peptide is weak and/or broken, reflecting weaker binding at this low pH, but the density of the imidazole is well defined enough to resolve two imidazole components. The Ndelta 1 atom of one component is not within hydrogen bonding distance of NGln and is surely protonated. Although the refined Ndelta 1His-NGln distance (2.92 ± 0.13 Å) of the other component suggests a hydrogen bond, the NGln-HGln-Ndelta 1His angle is poor (123 ± 5°), lower by 4.1 sigma  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 Ndelta 1His-NGln 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 I4122 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 I4122 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 I4122 complexes at pH values > 2.5 where the density is well defined, the temperature factors relatively low, and the Ndelta 1His-NGln distances and associated angles (Table III) clearly indicate an Ndelta 1His-NGln hydrogen bond at all three sites. However, at pH values <=  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-NH2, 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° about the Calpha -Cbeta bond with respect to the unprotonated conformer. The distance and angle parameters of the unprotonated conformer indicate an Ndelta 1His-NGln 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 pKa 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 pKa of 2.2. By contrast, at site 1, the occupancies of any unprotonated components at pH >=  2.5 are not high enough to observe or to resolve. Thus the pKa of the bound peptide His is detectably lower at site 1 than at site 2.

In the conformation of the protonated component at site 2 in I222 streptavidin-cyclo-[5-S-valeramide-HPQGPPC]K-NH2, pH 2.5, shown in Fig. 2B, the side chain is rotated 180 ° about the Cbeta -Cgamma bond with respect to the conformation of the unprotonated component to allow Ndelta 1 to make a hydrogen bond (shown in cyan) with a water molecule. This change in the His conformation increases the Nepsilon 2His-Ogamma Ser88 distance from 3.05 to 3.21 Å and decreases the Nepsilon 2His-Hepsilon 2His-Ogamma Ser88 angle from 163 to 108 °, indicating a weakening or loss of the hydrogen bond involving Nepsilon 2His and Ogamma Ser88. However an alternate hydrogen bond, Cepsilon 1His-Ogamma Ser88 = 2.98 Å, Cepsilon 1His-Hepsilon 1His-Ogamma Ser88 = 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-NH2 complex the average temperature factor of the imidazole at pH 2.5, 3.0, and 3.5 is lower at site 1 (21 ± 4 Å2) than at site 2 (36 ± 5 Å2), whereas at pH 1.5 it increases dramatically at both sites, becoming the same at site 1 (63 ± 2 Å2) as at site 2 (64 ± 2 Å2). For the I4122 complexes, the ligand temperature factors are as low as at site 1 in the I222 complexes.


DISCUSSION

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 pKa of ~6.3 in the unbound state involved in binding. The unperturbed pKa for the His side chain fully exposed to water is 6.3 (43). The observation of the Ndelta 1His-NGln 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 pKa 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 Ndelta 1His-NGln hydrogen bond are required for high affinity binding both in the crystals and in solution.

Table III provides the Ndelta 1His-NGln 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-CHPQGPPC-NH2, and linear Ac-CHPQFC-NH2.2 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 pKa 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]-NH2 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 2.2 In this investigation site 1 and site 2 were also shown to differ with respect to the protonation state of the His in bound HPQ-containing peptide ligands. The pKa of the peptide His is lower at the more solvent-shielded site 1 than at site 2. In the I4122 space group the pKa 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 pKa of the peptide His at site 1 in I222 streptavidin complexes and at the unique site in I4122 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 pKa of the peptide His at this site from the pKa in solution. Thus the crystallographically determined pKa 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 > 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 Nepsilon 2Gln and Ogamma 2Thr90 is directed right between the Gln Nepsilon 2 hydrogens, and there are two proton donors to Ogamma 2Thr90 (Nepsilon 2Gln and Nepsilon 1Trp79). In the more favorable orientation, the Ogamma 2 proton of Thr90 is directed at Oepsilon 1Gln, whereas one of the Nepsilon 2Gln hydrogens is directed at the oxygen of Wat600. In this arrangement Ogamma 2Thr90 receives a proton from Nepsilon 1Trp79 and donates a proton to Oepsilon 1Gln. 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 Ogamma 2Thr90 is ~90 °. The peptide Gln side chain is also uniquely oriented because of its interaction (d = 3.51 ± 0.15 Å, determined from 12 structures) with the Trp108 ring, which reflects an NH right-arrow pi  aromatic ring system hydrogen bond similar to those described (45).


Fig. 3. A, low energy hydrogen bonding network for Trp79, Thr90, a bound water, and the peptide Gln. B, alternate, higher energy, hydrogen bonding scheme for these groups.
[View Larger Version of this Image (22K GIF file)]

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 Odelta 1 atom and Ndelta 2Asn23. Asn23 is in turn uniquely oriented due to the hydrogen bond between its Odelta 1 atom and NSer27. The peptide Asn side chain is also uniquely oriented because of an Ndelta 2Asn23 right-arrow pi Trp120 interaction (d = 3.64 ± 0.04 Å, determined from four structures). Because the peptide His Ndelta 1 atom accepts a proton from the peptide Gln main chain NH group, the peptide His Nepsilon 2 atom must donate a proton to Ogamma Ser88. The two other peptide groups, OGln and NThr, 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.


Fig. 4. Hydrogen bonding network in streptavidin-FSHPQNT. Protein residues are white, peptide ligand is yellow-orange, and bound waters are light blue.
[View Larger Version of this Image (91K GIF file)]

Cyclic Peptides

Fig. 5 shows the hydrogen bonding interactions for streptavidin-cyclo-Ac-[CHPQFC]-NH2, pH 3.0. The directionalities of the hydrogen bond interactions involving this cyclic peptide are also unambiguous based on considerations similar to those described above for the streptavidin-bound linear peptide. The same hydrogen bonding network is observed at pH 11.8, 7.5, 4.8, 4.2, 3.6, and 2.0 with a protonated His component at the latter pH. Likewise, the hydrogen bond directionalities involving the binding of cyclo-Ac-[CHPQGPPC]-NH2 to streptavidin at pH 2.5, 2.9, 3.7, 5.0, and 7.5 are unambiguous (Fig. 6). The same network is observed in the complex with cyclo-[5-S-valeramide-HPQGPPC]K-NH2 at pH 1.5, 2.5, 3.0, and 3.5; cyclo-[5-S-valeramide-HPQGPPC]-NH2, pH 6.0 (22); cyclo-[4-S-toluamide-HPQGPPC]-NH2, pH 6.0 (22); head-to-head cyclo-[CHPQGPPC]-NH2 dimer, pH 2.5, 3.5, 5.0 (21), and 6.0 (21); and head-to-tail cyclo-[CHPQGPPC]-NH2 dimer, pH 2.5, 3.5, and 7.3 (19), with protonated peptide His components at pH values <=  2.5 in these complexes.


Fig. 5. Hydrogen bonding network in streptavidin-cyclo-Ac-[CHPQFC]-NH2. Protein residues are white, peptide ligand is yellow-orange, and bound waters are light blue.
[View Larger Version of this Image (89K GIF file)]


Fig. 6. Hydrogen bonding network in streptavidin-cyclo-Ac-[CHPQGPPC]-NH2. Protein residues are white, peptide ligand is yellow-orange, and bound waters are light blue. The peptide segments before and after the HPQG segment are schematic (atoms are missing).
[View Larger Version of this Image (93K GIF file)]

In a previous study of the binding of streptavidin to FSHPQNT, 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 streptavidin-cyclic peptide complexes at pH values > 2.5. In this investigation any disorder in the His occurs at low pH (< 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 pKa of the Peptide Histidine

The experimentally determined pKa values of certain noncatalytic residues in naturally occurring proteins are shifted by as much as 2.5 units (46). Larger shifts in the pKa values often occur in functionally important residues, such as those at active sites where buried charged residues often participate in catalysis (47-51). For example, the pKa of active site Asp26 in reduced thioredoxin is elevated by more than 5 units (52). The pKa values of charged residues engineered into hydrophobic cores of protein mutants are also perturbed by as much as 3.9 units (53-55). 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 pKa values. Large shifts in pKa values can also be effected simply by desolvation, the dominant factor shifting the pKa 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 (Ndelta 1His-NGln and Nepsilon 2His-Ogamma 2Thr90) at pH values >=  2.5. Thus, hydrogen bonding interactions together with desolvation within a protein cavity of low dielectric constant are among the factors perturbing the pKa in the streptavidin-HPQ-containing peptide complexes. Although the Cdelta 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 Trp79, Leu110, Ser88, Ala86, and Trp120 of a neighboring subunit. Upon protonation at low pH a small rotation about chi 1 of the peptide His and a 180 ° rotation about chi 2 allows Ndelta 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 pKa of the peptide His from long range crystal packing interactions should be taken as a caveat in extrapolation of pKa values determined in crystals to the corresponding ones in solution. The determination of the greatly reduced pKa of the His in streptavidin-bound HPQ-containing peptides and of the difference in pKa 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.


FOOTNOTES

*   The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    To whom correspondence should be addressed: Arris Pharmaceutical Corp., 385 Oyster Point Blvd., Suite 3, South San Francisco, CA 94080. Tel.: 415-829-1010; Fax: 415-829-1001; E-mail: bak{at}arris.com.
1   The abbreviations used are: CAPS, 3-[cyclohexylamino]-1-propanesulfonic acid; bis-Tris, [bis-(2-hydroxyethyl)-amino]-tris-(hydroxymethyl)-methane; Fo and Fc, observed and calculated structure factors; alpha c, calculated phases.
2   B. A. Katz, unpublished observations.

REFERENCES

  1. Green, N. M. (1975) Adv. Protein Chem. 29, 85-143 [Medline] [Order article via Infotrieve]
  2. Chilkoti, A., Schwartz, B. L., Smith, R. D., Long, C. J, and Stayton, P. S. (1995) Biotechnology 13, 1198-1204 [Medline] [Order article via Infotrieve]
  3. Green, N. M. (1990) Methods Enzymol. 184, 51-67 [Medline] [Order article via Infotrieve]
  4. Wilchek, M., and Bayer, E. A. (1990) Methods Enzymol. 184, 5-13 [Medline] [Order article via Infotrieve]
  5. Wilchek, M., and Bayer, E. A. (1990) Methods Enzymol. 184, 14-45 [Medline] [Order article via Infotrieve]
  6. Giebel, L. B., Cass, R. T., Milligan, D., Young, D., Arze, R., and Johnson, C. (1995) Biochemistry 34, 15430-15435 [Medline] [Order article via Infotrieve]
  7. Saggio, I., and Laufer, R. (1993) Biochem. J. 293, 613-616 [Medline] [Order article via Infotrieve]
  8. Kay, B. K., Adey, N. B., He, Y.-S., Manfredi, J. P., Mataragnon, A. H., and Fowlkes, D. M. (1993) Gene (Amst.) 128, 59-65 [CrossRef][Medline] [Order article via Infotrieve]
  9. Roberts, D., Gueglar, K., and Winter, J. (1993) Gene (Asmt.) 128, 67-69
  10. Lam, K. S., Salmon, S. E., Hersh, E. M., Hruby, V. J., Kazmierski, W. M., and Knapp, R. J. (1991) Nature 354, 82-84 [CrossRef][Medline] [Order article via Infotrieve]
  11. Devlin, J. J., Panganiban, L. C., and Devlin, P. E. (1990) Science 249, 404-406 [Medline] [Order article via Infotrieve]
  12. Katz, B. A., Liu, B., and Cass, R. (1996) J. Am. Chem. Soc. 118, 7914-7920 [CrossRef]
  13. Katz, B. A. (1995) Biochemistry 34, 15421-15429 [Medline] [Order article via Infotrieve]
  14. Weber, P. C., Wendoloski, J. J., Pantoliano, M. W., and Salemme, F. R. (1992) J. Am. Chem. Soc. 114, 3197-3200
  15. Hendrickson, W. A., Pähler, A., Smith, J. L., Satow, Y., Merritt, E. A., and Phizackerley, R. P. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 2190-2194 [Abstract]
  16. Weber, P. C., Ohlendorf, D. H., Wendoloski, J. J., and Salemme, F. R. (1989) Science 234, 85-88
  17. Sano, T., Pandori, M. W., Chen, X., Smith, C. L., and Cantor, C. R. (1995) J. Biol. Chem. 270, 28204-28209 [Abstract/Free Full Text]
  18. Reznik, G., Vajda, S., Smith, C. L., Cantor, C. R., and Sano, T. (1996) Nat. Biotechnol. 14, 1007-1011 [Medline] [Order article via Infotrieve]
  19. Katz, B. A. (1996) J. Am. Chem. Soc. 118, 2535-2536 [CrossRef]
  20. Katz, B. A., Cass, R. T., Liu, B., Arze, R., and Collins, N. (1995) J. Biol. Chem. 270, 31210-31218 [Abstract/Free Full Text]
  21. Katz, B. A., Stroud, R. M., Collins, N., Liu, B., and Arze, R. (1995) Chem. Biol. 2, 591-600 [Medline] [Order article via Infotrieve]
  22. Katz, B. A., Johnson, C., and Cass, R. T. (1995) J. Am. Chem. Soc. 117, 8541-8547
  23. Weber, P. C., Pantoliano, M. W., Simons, D. M., and Salemme, F. R. (1994) J. Am. Chem. Soc. 116, 2717-2724
  24. Green, N. M. (1966) Biochem. J. 101, 774-780
  25. Weber, P. C., Pantoliano, M. W., and Thompson, L. D. (1992) Biochemistry 31, 9350-9354 [Medline] [Order article via Infotrieve]
  26. Zheng, R.-L., and Kemp, R. G. (1994) J. Biol. Chem. 269, 18475-18479 [Abstract/Free Full Text]
  27. Qamar, R., and Cook, P. F. (1993) Biochemistry 32, 6802-6806 [Medline] [Order article via Infotrieve]
  28. Knowles, J. R. (1976) CRC Crit. Rev. Biochem. 4, 165-173 [Medline] [Order article via Infotrieve]
  29. Swint-Kruse, L., and Robertson, A. D. (1996) Biochemistry 35, 171-180 [CrossRef][Medline] [Order article via Infotrieve]
  30. Raghavan, M., Bonagura, V. R., Morrison, S. L., and Bjorkman, P. J. (1995) Biochemistry 34, 14649-14657 [Medline] [Order article via Infotrieve]
  31. D'Souza, U. M., and Strange, P. G. (1995) Biochemistry 34, 13635-13641 [Medline] [Order article via Infotrieve]
  32. Cheng, Y., Mason, A. B., and Woodworth, R. C. (1995) Biochemistry 34, 14879-14884 [Medline] [Order article via Infotrieve]
  33. Persson, E., Ezban, M., and Shymko, R. M. (1995) Biochemistry 34, 12775-12781 [Medline] [Order article via Infotrieve]
  34. Huang, S. G., and Klingenberg, M. (1995) Biochemistry 34, 349-360 [Medline] [Order article via Infotrieve]
  35. Fay, S. P., Habbersett, R., Domalewski, M. D., Posner, R. G., Houghton, T. G., Pierson, E., Muthukumaraswamy, N., Whitaker, J., Haughland, R. P., Freer, R. J., and Sklar, L. A. (1994) Cytometry 15, 148-153 [Medline] [Order article via Infotrieve]
  36. Boniface, J. J., Allbritton, N. L., Reay, P. A., Kantor, R. M., Stryer, L., and Davis, M. M. (1993) Biochemistry 32, 11761-11768 [Medline] [Order article via Infotrieve]
  37. Pähler, A., Hendrickson, W. A., Kolks, M. A. G., Argaraña, C. E., and Cantor, C. R. (1987) J. Biol. Chem. 262, 13933-13937 [Abstract/Free Full Text]
  38. Brünger, A. T. (1992) Xplor Version 3.1: A System for X-ray Crystallography and NMR, pp. 187-206, Yale University Press, New Haven, CT
  39. Chambers, J. L., and Stroud, R. M. (1979) Acta Crystallogr. Sec. B 33, 1861-1871
  40. Finer-Moore, J. S., Kossiakoff, A. A., Hurley, J. H., Earnest, T., and Stroud, R. M. (1992) Proteins 12, 203-222 [Medline] [Order article via Infotrieve]
  41. Karlsson, R., and Stahlberg, R. (1995) Anal. Biochem. 228, 274-280 [CrossRef][Medline] [Order article via Infotrieve]
  42. Derewenda, Z. S., Lee, L., and Derewenda, U. (1995) J. Mol. Biol. 252, 248-262 [CrossRef][Medline] [Order article via Infotrieve]
  43. Antosiewicz, J., McCammon, J. A., and Gilson, M. K. (1994) J. Mol. Biol. 238, 415-436 [CrossRef][Medline] [Order article via Infotrieve]
  44. McDonald, I. K., and Thornton, J. M. (1994) Protein Eng. 8, 217-224 [Abstract]
  45. Burley, S. K., and Petsko, G. A. (1986) FEBS Lett. 203, 139-143 [CrossRef][Medline] [Order article via Infotrieve]
  46. Bashford, D., and Karplus, M. (1990) Biochemistry 9, 327-335
  47. Kossiakoff, A. A. (1983) Annu. Rev. Biophys. Bioeng. 12, 159-182 [Medline] [Order article via Infotrieve]
  48. Baldwin, J., and Chothia, C. (1979) J. Mol. Biol. 129, 175-220 [Medline] [Order article via Infotrieve]
  49. Gelin, B. R., and Karplus, M. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 801-805 [Abstract]
  50. Perutz, M. F. (1978) Science 201, 1187-1191 [Medline] [Order article via Infotrieve]
  51. Kraut, J. (1977) Annu. Rev. Biochem. 46, 331-358 [CrossRef][Medline] [Order article via Infotrieve]
  52. Wilson, N. A., Barbar, E., Fuchs, J. A., and Woodward, C. (1995) Biochemistry 34, 8931-8939 [Medline] [Order article via Infotrieve]
  53. Stites, W. E., Gittis, A. G., Lattman, E. E., and Shortle, D. (1991) J. Mol. Biol. 221, 7-14 [CrossRef][Medline] [Order article via Infotrieve]
  54. Dao-pin, S., Anderson, D. E., Baase, W. A., Dahlquist, F. W., and Matthews, B. W. (1991) Biochemistry 30, 11521-11529 [Medline] [Order article via Infotrieve]
  55. Varadarajan, R., Lambright, D. G., and Boxer, S. G. (1989) Biochemistry 28, 3771-3781 [Medline] [Order article via Infotrieve]
  56. Antosiewicz, J., and McCammon, J. A. (1996) Biochemistry 35, 7819-7833 [CrossRef][Medline] [Order article via Infotrieve]
  57. Gilson, M. K. (1995) Current Opin. Struct. Biol. 5, 216-223 [CrossRef]
  58. Konig, B., and Nichols, A. (1995) Science 268, 1144-1149 [Medline] [Order article via Infotrieve]
  59. Warshel, A., and Russel, S. (1984) Q. Rev. Biophys. 17, 283-422 [Medline] [Order article via Infotrieve]
  60. Urry, D. W., Gowda, D. C., Peng, S., Parker, T. M., Jing, N., and Harris, R. D. (1994) Biopolymers 34, 889-896 [Medline] [Order article via Infotrieve]
  61. Rossmann, M. G., Leslie, A. G. W., Abdel-Meguid, S. S., and Tsukihara, T. (1979) J. Appl. Crystallogr. 12, 570-581 [CrossRef]
  62. Brünger, A. T. (1992) Nature 355, 472-474 [CrossRef]

©1997 by The American Society for Biochemistry and Molecular Biology, Inc.