Thermodynamics of unfolding for Kazal-type serine protease inhibitors: entropic stabilization of ovomucoid first domain by glycosylation.

A synthetic gene for chicken ovomucoid first domain (OMCHI1) has been overexpressed in Escherichia coli. The resulting recombinant protein, rOMCHI1, is expressed and correctly folded without the use of fusion proteins or export secretion signal peptides incorporated into the gene. The thermostability of rOMCHI1 has been compared to that of the naturally occurring glycosylated OMCHI1 (gOMCHI1). The results of differential scanning calorimetry (DSC) studies show that the heat capacity change for unfolding, deltaCp, for both rOMCHI1 and gOMCHI1 is approximately 600 cal/(mol x K). At any given pH, however, the presence of N-linked carbohydrate increases the Tm for thermal unfolding of gOMCHI1 over rOMCHI1 by 2-4 degrees C, without changing the enthalpy of unfolding, delta H(degree)m. This suggests that the increased thermal stability of gOMCHI1 is entropic. Comparison of the unfolding thermodynamics of rOMCHI1 with those of turkey ovomucoid third domain (OMTKY3), which is 36% identical to rOMCHI1, reveals similar deltaCp values for both proteins, about 600 cal/(mol x K), but a reduction in delta H(degree)m of about 5 kcal/mol for rOMCHI1 at all temperatures. Decreases in delta H(degree)m for rOMCHI1 versus OMTKY3 may be explained by an overall less ordered native state in rOMCHI1. In the absence of a native structure for OMCHI1, the change in accessible surface area upon unfolding, deltaASA, was calculated using unfolding parameters and structural energetic relationships [Murphy & Freire (1992) Adv. Protein Chem. 43, 313-361; Murphy et al. (1993), Proteins: Struct., Funct., Genet. 15, 113-120]. These calculations suggest that the larger protein rOMCHI1 (Mr 7500) exposes less surface area than OMTKY3 (Mr 6100) upon thermal denaturation. Overall, structural energetic relationships may provide a useful framework for interpretation and comparison of thermodynamic data for structurally homologous proteins.

Molecular bases for human leucocyte elastase inhibition.

Human leucocyte elastase is a serine proteinase involved in phagocytosis, defence against invading micro-organisms, degradation of elastin, collagen, proteoglycans, fibrinogen and fibrin, being also responsible for the digestion of damaged tissues and of the bacterial degradation products. Lack of the enzyme regulation is at the basis of pathological states, such as pulmonary emphysema, cystic fibrosis, rheumatoid arthritis, atherosclerosis and glomerulonephritis. A detailed characterisation of the enzyme:inhibitor recognition process, based on extensive thermodynamic, kinetic and structural information, as well as on the comparative analysis with the homologous proteinase from porcine pancreas, is reported in the present review.

Molecular skins: a new concept for quantitative shape matching of a protein with its small molecule mimics.

A novel analytical method for comparing molecular shapes by optimizing the intersection of molecular "SKINS" has been developed. This method provides a quantitative measure of the shape similarity by maximizing the intersection volume of molecular surfaces with a finite thickness; a molecular skin. We report shape matching of a small tripeptide inhibitor (DFKi) of elastase class proteins with the 56 residue turkey ovomucoid inhibitor (TOMI). To match a large elastase inhibitor such as TOMI with a small inhibitor or drug, we found that it is necessary to use a skin match rather than molecular volume. Skin based comparisons of TOMI protein with DFKi successfully found the alignment expected from comparison of their respective crystallographic complexes with elastase (i.e. HLE/TOMI complex and PPE/tripeptide complex). In the skin comparison of the tripeptide with the TOMI protein, blind searching for skin matches involved optimization of the skin intersection from 172 starting positions randomly selected from a set of 500 points on the TOMI van der Waals surface [within 9.5 A of the Leu-18 on the TOMI binding loop (1 point/A2)]. The tripeptide center of mass was placed at these points and its orientation was randomized before optimization was initiated. The best skin intersection, 86.4 A3, was found three times and corresponds to the experimental alignment. The next best skin intersection was 78.1 A3 giving a discrimination factor in this case of 10%. Searches over the entire surface of the TOMI protein did not identify any new matches with skin intersection greater than 78.1 A3. Matching the DFKi with a TOMI structure relaxed from its crystal conformation by molecular dynamics gives similar results.

Deglycosylation with trifluoromethanesulfonic acid differentially affects inhibitor activities of turkey ovomucoid.

Turkey ovomucoid is an inhibitor of both trypsin and chymotrypsin. Treatment of this glycoprotein with trifluoromethanesulfonic acid in anisole resulted in time-dependent removal of carbohydrate and altered its biological activity. After 6 h of treatment the apparent molecular mass obtained by SDS-PAGE decreased from 38 to 30 kDa. Carbohydrate analyses indicated loss of 94% of original saccharide residues. The inhibitory activity of each domain was analyzed independently by comparing enzymic activity of trypsin and chymotrypsin in the absence of inhibitor to that preincubated in the presence of varying amounts of native or deglycosylated ovomucoid, respectively. The results demonstrated that removal of saccharides with trifluoromethanesulfonic acid differentially affects the inhibitor activities of turkey ovomucoid. Decreased inhibitory activity of the trypsin domain was observed with casein and benzoyl arginine ethyl ester as substrates. In contrast, enhanced inhibitory activity of the chymotrypsin domain was observed with benzoyl tyrosine ethyl ester and methyl-O-succinyl-Arg-Pro-Tyr-p-nitroanilide, good substrates for chymotrypsin, but not with casein.

Modularity of protein function: chimeric interleukin 1 beta s containing specific protease inhibitor loops retain function of both molecules.

Although it is widely recognized that many proteins contain discrete functional domains, it is less certain whether smaller, less obviously discrete, units of structure will retain their specific function when transplanted into a different context. The observation that the potent inflammatory cytokine human interleukin 1 beta has the same overall structure as soybean trypsin inhibitor (STI) (Kunitz) prompted us to replace a tight turn in the cytokine sequence with the large loop in soybean trypsin inhibitor that binds to the active site of trypsin. Wild-type interleukin 1 beta (IL-1 beta) is highly resistant to proteolysis, but the chimeric STI/IL is specifically cleaved by trypsin, apparently in the inserted loop. Other chimeric interleukins have also been constructed, by replacing the same tight turn with inhibitory loops from other protein protease inhibitors: turkey ovomucoid inhibitor (TOI), a chymotrypsin inhibitor, and alpha 1-antitrypsin (AT), an elastase inhibitor. Although these loops come from proteins not related structurally to interleukin 1, they confer specific protease sensitivity or inhibition on the chimeric cytokine. The cytokine properties of these chimeric interleukins have also been evaluated. The chimeras formed from human IL-1 beta and all inhibitory loops tested bind to the interleukin 1 receptor with reasonable affinity. The typical cellular effects of IL-1, however, are not observed with all the recombinant proteins, thus confirming that receptor binding and signal transduction can be uncoupled. When these results are taken together with the results of site-directed mutagenesis of IL-1, reported in this paper and elsewhere, they allow the receptor and intracellular transduction sites on the protein to be mapped in detail.

Refined X-ray crystal structures of the reactive site modified ovomucoid inhibitor third domains from silver pheasant (OMSVP3*) and from Japanese quail (OMJPQ3*).

Tetragonal and triclinic crystals of two ovomucoid inhibitor third domains from silver pheasant and Japanese quail, modified at their reactive site bonds Met18-Glu19 (OMSVP3*) and Lys18-Asp19 (OMJPQ3*), respectively, were obtained. Their molecular and crystal structures were solved using X-ray data to 2.5 A and 1.55 A by means of Patterson search methods using truncated models of the intact (virgin) inhibitors as search models. Both structures were crystallographically refined to R-values of 0.185 and 0.192, respectively, applying an energy restraint reciprocal space refinement procedure. Both modified inhibitors show large deviations from the intact derivatives only in the proteinase binding loops (Pro14 to Arg21) and in the amino-terminal segments (Leu1 to Val6). In the modified inhibitors the residues immediately adjacent to the cleavage site (in particular P2, P1, P1') are mobile and able to adapt to varying crystal environments. The charged end-groups, i.e. Met18 COO- and Glu19 NH3+ in OMSVP3*, and Lys18 COO- and Asp19 NH3+ in OMJPQ3*, do not form ion pairs with one another. The hydrogen bond connecting the side-chains of Thr17 and Glu19 (i.e. residues on either side of the scissile peptide bond) in OMSVP3 is broken in the modified form, and the hydrogen-bond interactions observed in the intact molecules between the Asn33 side-chain and the carbonyl groups of loop residues P2 and P1' are absent or weak in the modified inhibitors. The reactive site cleavage, however, has little effect on specific interactions within the protein scaffold such as the side-chain hydrogen bond between Asp27 and Tyr31 or the side-chain stacking of Tyr20 and Pro22. The conformational differences in the amino-terminal segment Leu1 to Val6 are explained by their ability to move freely, either to associate with segments of symmetry-related molecules under formation of a four-stranded beta-barrel (OMSVP3* and OMJPQ3) or to bind to surrounding molecules. Together with the results given in the accompanying paper, these findings probably explain why Khyd of small protein inhibitors of serine proteinases is generally found to be so small.

Deoxyribonuclease I sensitivity of the ovomucoid-ovoinhibitor gene complex in oviduct nuclei and relative location of CR1 repetitive sequences.

The location of CR1 middle repetitive sequences within or near the boundaries of the ovalbumin DNase I sensitive domain has suggested that CR1 sequences may play a role in defining transition regions of DNase I sensitivity in hen oviduct nuclei. We have examined this apparent relationship of CR1 sequences and transitions of chromatin structure by determining the DNase I sensitivity in oviduct nuclei of a 47-kilobase region that contains five CR1 sequences and the transcribed ovomucoid and ovoinhibitor genes. We find that three of the CR1 sequences occur within a broad transition region of decreasing DNase I sensitivity downstream of the ovomucoid gene. Another CR1 is in a region of decreased DNase I sensitivity within the ovoinhibitor gene. The fifth CR1 sequence is in a DNase I sensitive region between the two genes but which is less sensitive to DNase I digestion than the region immediately upstream from the ovomucoid gene. Thus, the CR1 sequences occur within regions of reduced relative DNase I sensitivity, suggesting that CR1s could facilitate the formation of a chromatin conformation that is less sensitive to DNase I digestion. Unexpectedly, the noncoding strand of sequences within and immediately adjacent to the 5' end of the actively transcribed ovomucoid and ovalbumin genes was less sensitive to DNase I digestion than their respective coding strands.

The crystal and molecular structure of the third domain of silver pheasant ovomucoid (OMSVP3).

OMSVP3 and OMTKY3 (third domains of silver pheasant and turkey ovomucoid inhibitor) are Kazal-type serine proteinase inhibitors. They have been isomorphously crystallized in the monoclinic space group C2 with cell dimensions of a = 4.429 nm, b = 2.115 nm, c = 4.405 nm, beta = 107 degrees. The asymmetric unit contains one molecule corresponding to an extremely low volume per unit molecular mass of 0.0017 nm3/Da. Data collection was only possible for the OMSVP3 crystals. Orientation and position of the OMSVP3 molecules in the monoclinic unit cells were determined using Patterson search methods and the known structure of the third domain of Japanese quail ovomucoid (OMJPQ3) [Papamokos, E., Weber, E., Bode, W., Huber, R., Empie, M. W., Kato, I. and Laskowski, M., Jr (1982) J. Mol. Biol. 158, 515-537]. The OMSVP3 structure has been refined by restrained crystallographic refinement yielding a final R value of 0.199 for data to 0.15 nm resolution. Conformation and hydrogen-bonding pattern of OMSVP3 and OMJPQ3 are very similar. Large deviations occur at the NH2 terminus owing to different crystal packing, and at the C terminus of the central helix, representing an intrinsic property and resulting from amino acid substitutions far away from this site. The deviation of OMSVP3 from OMTKY3 complexed with the Streptomyces griseus protease B is very small [Fujinaga, M., Read, R. J., Sielecki, A., Ardelt, W., Laskowski, M., Jr and James, M. N. G. (1982) Proc. Natl Acad. Sci. USA, 79, 4868-4872].