Daniel Krowarsch

Canonical protein inhibitors of serine proteases.

Serine proteases and their natural protein inhibitors are among the most intensively studied protein complexes. About 20 structurally diverse inhibitor families have been identified, comprising α-helical, β sheet, and α/β proteins, and different folds of small disulfide-rich proteins. Three different types of inhibitors can be distinguished based on their mechanism of action: canonical (standard mechanism) and non-canonical inhibitors, and serpins. The canonical inhibitors bind to the enzyme through an exposed convex binding loop, which is complementary to the active site of the enzyme. The mechanism of inhibition in this group is always very similar and resembles that of an ideal substrate. The non-canonical inhibitors interact through their N-terminal segment. There are also extensive secondary interactions outside the active site, contributing significantly to the strength, speed, and specificity of recognition. Serpins, similarly to the canonical inhibitors, interact with their target proteases in a substrate-like manner; however, cleavage of a single peptide bond in the binding loop leads to dramatic structural changes.

The impact of Glu→Ala and Glu→Asp mutations on the crystallization properties of RhoGDI: the structure of RhoGDI at 1.3 Å resolution

It is hypothesized that surface residues with high conformational entropy, specifically lysines and glutamates, impede protein crystallization. In a previous study using a model system of Rho-specific guanine nucleotide dissociation inhibitor (RhoGDI), it was shown that mutating Lys residues to Ala results in enhanced crystallizability, particularly when clusters of lysines are targeted. It was also shown that one of these mutants formed crystals that yielded diffraction to 2.0 A, a significant improvement on the wild-type protein crystals. In the current paper, an analysis of the impact of surface mutations replacing Glu residues with Ala or Asp on the stability and crystallization properties of RhoGDI is presented. The Glu-->Ala (Asp) mutants are generally more likely to produce crystals of the protein than the wild-type and in one case the resulting crystals yielded a diffraction pattern to 1.2 A resolution. This occurs in spite of the fact that mutating surface Glu residues almost invariably affects the proteins stability, as illustrated by the reduced deltaG between folded and unfolded forms measured by isothermal equilibrium denaturation. The present study strongly supports the notion that rational surface mutagenesis can be an effective tool in overcoming problems stemming from the proteins recalcitrance to crystallization and may also yield dramatic improvements in crystal quality.

Increased protein stability of FGF1 can compensate for its reduced affinity for heparin

Human FGF1 (fibroblast growth factor 1) is a powerful signaling molecule with a short half-life in vivo and a denaturation temperature close to physiological. Binding to heparin increases the stability of FGF1 and is believed to be important in the formation of FGF1·fibroblast growth factor receptor (FGFR) active complex. In order to reveal the function of heparin in FGF1·FGFR complex formation and signaling, we constructed several FGF1 variants with reduced affinity for heparin and with diverse stability. We determined their biophysical properties and biological activities as well as their ability to translocate across cellular membranes. Our study showed that increased thermodynamic stability of FGF1 nicely compensates for decreased binding of heparin in FGFR activation, induction of DNA synthesis, and cell proliferation. By stepwise introduction of stabilizing mutations into the K118E (K132E) FGF1 variant that shows reduced affinity for heparin and is inactive in stimulation of DNA synthesis, we were able to restore the full mitogenic activity of this mutant. Our results indicate that the main role of heparin in FGF-induced signaling is to protect this naturally unstable protein against heat and/or proteolytic degradation and that heparin is not essential for a direct FGF1-FGFR interaction and receptor activation.

The impact of Lys→Arg surface mutations on the crystallization of the globular domain of RhoGDI

The potential of rational surface mutagenesis for enhanced protein crystallization is being probed in an ongoing effort. In previous work, it was hypothesized that residues with high conformational entropy such as Glu and Lys are suitable targets for surface mutagenesis, as they are rarely incorporated in crystal contacts or protein-protein interfaces. Previous experiments using Lys-->Ala, Glu-->Ala and Glu-->Asp mutants confirmed that mutated proteins were more likely to crystallize. In the present paper, the usefulness of Lys-->Arg mutations is studied. Several mutations of the globular domain of human RhoGDI were generated, including the single mutants K105R, K113R, K127R, K138R and K141R, the double mutants K(98,99)R and K(199,200)R and the triple mutants K(98,99,105)R and K(135,138,141)R. It is shown that Lys-->Arg mutants are more likely to crystallize than the wild-type protein, although not as likely as Lys-->Ala mutants. Out of the nine mutants tested, five produced diffracting crystals, including the K(199,200)R double mutant, which crystallized in a new space group and exceeded by approximately 1.0 A the resolution of the diffraction of the wild-type crystal. Major crystal contacts in the new lattice were created by the mutated epitope.

Analysis of serine proteinase-inhibitor interaction by alanine shaving.

We analyzed the energetic importance of residues surrounding the hot spot (the P1 position) of bovine pancreatic trypsin inhibitor (BPTI) in interaction with two proteinases, trypsin and chymotrypsin, by a procedure called molecular shaving. One to eight residues of the structural epitope, composed of two extended and exposed loops, were mutated to alanine(s). Although truncation of the side chains of residues surrounding the P1 position to methyl groups caused a decrease in ΔGden values up to 6.4 kcal mole−1, it did not influence the overall conformation of the inhibitor. We found that the replacement of up to six residues with alanines was fully additive at the level of protein stability. To analyze the influence of the structural epitope on the association energy, we determined association constants for BPTI variants and both enzymes and applied the additivity analysis. Shaving of two binding loops led to a progressive drop in the association energy, more pronounced for trypsin (decrease up to 9.6 kcal mole−1) than chymotrypsin (decrease up to 3.5 kcal mole−1). In the case of extensively mutated variants interacting with chymotrypsin, the association energies agreed very well with the values calculated from single mutational effects. However, when P1‐neighboring residues were shaved to alanine(s), their contribution to the association energy was not fully removed because of the presence of methyl groups and main chain–main chain intermolecular hydrogen bonds. Moreover, the hot spot had a different contribution to the complex stability in the fully shaved BPTI variant compared with the wild type, which was caused by perturbations of the P1–S1 electrostatic interaction.

Thermodynamics of single peptide bond cleavage in bovine pancreatic trypsin inhibitor (BPTI)

A major goal of this paper was to estimate a dynamic range of equilibrium constant for the opening of a single peptide bond in a model protein, bovine pancreatic trypsin inhibitor (BPTI). Ten mutants of BPTI containing a single Xaa→Met substitution introduced in different parts of the molecule were expressed in Escherichia coli. The mutants were folded, purified to homogeneity, and cleaved with cyanogen bromide to respective cleaved forms. Conformation of the intact mutants was similar to the wildtype, as judged from their circular dichroism spectra. Substantial conformational changes were observed on the chemical cleavage of three single peptide bonds—Met46‐Ser, Met49‐Cys, and Met53‐Thr—located within the C‐terminal helix. Cleavage of those peptide bonds caused a significant destabilization of the molecule, with a drop of the denaturation temperature by 56.4°C to 68°C at pH 4.3. Opening of the remaining seven peptide bonds was related to a 10.8°C to 39.4°C decrease in Tden. Free energies of the opening of 10 single peptide bonds in native mutants (ΔGop,N) were estimated from the thermodynamic cycle that links denaturation and cleavage free energies. To calculate those values, we assumed that the free energy of opening of a single peptide bond in the denatured state (ΔGop,D) was equal to −2.7 kcal/mole, as reported previously. Calculated ΔGop,N values in BPTI were in the range from 0.2 to 10 kcal/mole, which was equivalent to a >1 million–fold difference in equilibrium constants. The values of ΔGop,N were the largest for peptide bonds located in the C‐terminal helix and significantly lower for peptide bonds in the β‐structure or loop regions. It appears that opening constants for single peptide bonds in various proteins span across 33 orders of magnitude. Typical equilibrium values for a single peptide bond opening in a protein containing secondary structure elements fall into negligibly low values, from 10−3 to 10−8, and are efficient to ensure stability against proteolysis.

Amino-acid substitutions at the fully exposed P1 site of bovine pancreatic trypsin inhibitor affect its stability

It is widely accepted that solvent‐exposed sites in proteins play only a neglible role in determining protein energetics. In this paper we show that amino acid substitutions at the fully exposed Lys15 in bovine pancreatic trypsin inhibitor (BPTI) influenced the CD‐ and DSC‐monitored stability: The Tden difference between the least (P1 Trp) and the most stable (P1 His) mutant is 11.2°C at pH 2.0. The ΔHden versus Tden plot for all the variants at three pH values (2.0, 2.5, 3.0) is linear (ΔCp,den = 0.41 kcal• mole−1 • K−1; 1 cal = 4.18 J) leading to a ΔGden difference of 2.1 kcal•mole−1. Thermal denaturation of the variants monitored by CD signal at pH 2.0 in the presence of 6 M GdmCl again showed differences in their stability, albeit somewhat smaller (ΔTden =7.1°C). Selective reduction of the Cys14–Cys 38 disulfide bond, which is located in the vicinity of the P1 position did not eliminate the stability differences. A correlation analysis of the P1 stability with different properties of amino acids suggests that two mechanisms may be responsible for the observed stability differences: the reverse hydrophobic effect and amino acid propensities to occur in nonoptimal dihedral angles adopted by the P1 position. The former effect operates at the denatured state level and causes a drop in protein stability for hydrophobic side chains, due to their decreased exposure upon denaturation. The latter factor influences the native state energetics and results from intrinsic properties of amino acids in a way similar to those observed for secondary structure propensities. In conclusion, our results suggest that the protein‐stability‐derived secondary structure propensity scales should be taken with more caution.

Biophysical Properties of the Eukaryotic Ribosomal Stalk

The landing platform for the translational GTPases is located on the 60S ribosomal subunit and is referred to as a GTPase-associated center. The most distinctive feature of this center is an oligomeric complex, the stalk, responsible for the recruitment of translation factors and stimulation of translation factor-dependent GTP hydrolysis. In eukaryotes, the stalk has been investigated in vitro and in vivo, but most information available concerns its individual components only. In the present study, we provide an insight into the biophysical nature of the native stalk isolated from the yeast Saccharomyces cerevisiae. Using fluorescence, circular dichroism, and mass spectrometry analyses, we were able to characterize the natively formed yeast stalk, casting new light on the oligomeric properties of the complex and its quaternary topology, showing that folding and assembly are coupled processes. The pentameric stalk is an exceptionally stable structure with the protein core composed of P0, P1A, and P2B proteins and less tightly bound P1B and P2A capable of dissociating from the stalk core. We obtained also the whole picture of the posttranslational modifications at the logarithmic phase of yeast growth, using mass spectrometry approach, where P proteins are phosphorylated at a single serine residue, P0 may accept two phosphate groups, and P1A none. Additionally, only P1B undergoes N-terminal acetylation after prior methionine removal.

Structure-Function Relationships in Serine Protease-Bovine Pancreatic Trypsin Inhibitor Interaction

We report our progress in understanding the structure-function relationships for the interaction between BPTI and serine proteases. We focused on extensive mutagenesis of four crucial positions from the protease binding loop of BPTI. Selected variants were characterized by determination of association constants, stability parameters and structures of protease-inhibitor complexes.

Synthesis, cloning and expression in Escherichia coli of a gene coding for the Met8 → Leu CMTI I — a representative of the squash inhibitors of serine proteinases

A chemically synthesized gene coding for a Cucurbita maxima trypsin inhibitor modified at position P′3 (Met8→ Leu CMTI I), i.e. at the third position downstream of the reactive site bond (Arg5‐Ile), was cloned into a derivative of the plasmid pAED4 that utilizes a T7 expression system. The gene was expressed in Escherichia coli as a fusion protein that accumulates in inclusion bodies. After reduction and CNBr cleavage of the fusion protein followed by oxidative refolding and reverse‐phase HPLC, about 5 mg of pure protein was obtained per 1 of cell culture. Association constants of recombinant Leu‐8‐CMTI I with bovine β‐trypsin and human cathepsin G are the same, within experimental error, as for CMTI I isolated from a natural source.