Paired Spectroscopic and Crystallographic Studies of Proteases

Abstract

The active sites of subtilisin and trypsin have been studied by paired IR spectroscopic and X-ray crystallographic studies. The active site serines of the proteases were reacted with 4-cyanobenzenesulfonyl fluoride (CBSF), an inhibitor that contains a nitrile vibrational reporter. The nitrile stretch vibration of the water-soluble inhibitor model, potassium 4-cyanobenzenesulfonate (KCBSO), and the inhibitor were calibrated by IR solvent studies in H₂O/DMSO and the frequency-temperature line-slope (FTLS) method in H₂O and THF. The inhibitor complexes were examined by FTLS and the slopes of the best fit lines for subtilisin-CBS and trypsin-CBS in aqueous buffer were both measured to be -3.5×10^-2 cm^-1/°C. These slopes were intermediate in value between that of KCBSO in aqueous buffer and CBSF in THF, which suggests that the active-site nitriles in both proteases are mostly solvated. The X-ray crystal structures of the subtilisin-CBS and trypsin-CBS complexes were solved at 1.27 and 1.32 Å, respectively. The inhibitor was modelled in two conformations in subtilisin-CBS and in one conformation in the trypsin-CBS. The crystallographic data support the FTLS data that the active-site nitrile groups are mostly solvated and participate in hydrogen bonds with water molecules. The combination of IR spectroscopy utilizing vibrational reporters paired with X-ray crystallography provides a powerful approach to studying protein structure.

Keywords: crystallography; proteases; spectroscopy; vibrational reporters.

Figure 1.

Structure of the inhibitor, 4-cyanobenzenesulfonyl fluoride (1) and potassium 4-cyanobenzenesulfonate (2).

Figure 2.

FTIR spectra of KCBSO and K¹³CBSO dissolved in water at a concentration of 250 mM in the region 2140 – 2280 cm^–1 recorded at 25 °C. The spectra were intensity normalized and baseline corrected.

Figure 3.

FTIR spectra of KCBSO in DMSO and H₂O mixtures (v/v) at a concentration of 250 mM in the region 2200 – 2270 cm^–1 recorded at 25 °C. The spectra were intensity normalized and baseline corrected.

Figure 4.

FTIR spectra of CBS bound to subtilisin (A, open circles) or trypsin (B, open squares) dissolved in an aqueous buffer (10 mM Hepes, 20 mM KCl, pH 7.5) in the region 2210 – 2270 cm^–1. The protein concentration was 2.5 mM. The spectra were recorded at 25 °C, intensity normalized, baseline corrected, and fit to a linear combination of a Gaussian and Lorentzian function (solid curves).

Figure 5.

Temperature dependent shifts in the nitrile stretching frequency of CBSF dissolved in THF (open circles) or KCBSO dissolved in an aqueous buffer consisting of 10 mM Hepes and 20 mM KCl at a pH of 7.5 (open squares). The temperature-dependent frequency shifts were fit to a straight line.

Figure 6.

Temperature dependent shifts in the nitrile stretching frequency for CBS bound to subtilisin (open circles) dissolved in an aqueous buffer (10 mM Hepes, 20 mM KCl, pH 7.5). The protein concentration was 2.5 mM. The temperature-dependent frequency shifts were fit to a straight line with a slope of −3.5±0.5×10⁻² cm⁻¹/°C.

Figure 7.

Crystal structure of subtilisin-CBS complex. A) subtilisin ribbon structure shown in pink with CBS inhibitor shown in sticks modeled in two conformations at the active site serine 220. B) Zoom in on active site in same orientation as A with modelled CBS inhibitors shown in sticks along with 2F_O - F_C electron density at 1σ shown in blue mesh, water shown as red sphere, and partially occupied calcium shown as green sphere C) Approximately 90° rotation from A/B orientation with protein surface shown in pink and CBS inhibitor shown in sticks.

Figure 8.

Comparison of various subtilisin structures. A) Ribbon structure alignment of subtilisin-CBS in pink, wild-type subtilisin from Bacillus Lentus in yellow (PDB ID: 1NDQ, RMSD 0.515Å), subtilisin-PMS in cyan (PDB ID: 3VYV, RMSD 0.306Å), and subtilisin-vinylPMS in grey (PDB ID: 5AQE, RMSD 0.476Å). B) Active site alignment show in sticks for subtilisin-CBS in pink, wild-type subtilisin in yellow, subtilisin-PMS in cyan, and subtilisin-vinylPMS in grey.

Figure 9.

Crystal structure of trypsin-CBS complex. A) Trypsin ribbon structure shown in green with CBS inhibitor shown in sticks modelled at the active site serine 200. B) Zoom in on active site in same orientation as A with modelled CBS inhibitor shown in sticks along unreacted serine with 2F_O - F_C electron density at 1σ shown in blue mesh. C) Approximately 90° rotation from A/B orientation with protein surface shown in green and CBS inhibitor shown in sticks.

Figure 10.

Comparison of various trypsin structures. A) Ribbon structure alignment of trypsin-CBS in green, trypsin-benzamide in yellow (PDB ID: 4I8H, RMSD 0.141Å), trypsin-PMS in cyan (PDB ID: 1PQA, RMSD 0.810Å), and trypsin-benzeneboronic acid in magenta (PDB ID: 2A32, RMSD 0.319Å). B) Active site alignment show in sticks for trypsin-CBS in green, trypsin-benzamide in yellow, trypsin-PMS in cyan, and trypsin-benzeneboronic acid in magenta.

Scheme 1.

Synthesis of K CBSO and K ¹³CBSO using copper (I) cyanide.