Greglin is a non-classical Kazal inhibitor with an extra N-terminal domain bearing an unusual combination of post-translational modifications
Greglin is an inhibitor composed of two distinct domains consisting of residues 1–20 and 21–83. At present, the function of the first domain (1–20), which does not possess inhibitory properties, remains unknown. The absence of this domain in the determined crystal structure correlates with its sensitivity to proteolysis, as previously reported . However, this domain displays a combination of post-translational modifications detected by mass spectrometry, i.e. three phosphorylations and a probable glycosylation. This situation is not commonly found in Kazal inhibitors, and may be linked to a specific biological activity, such as control of the formation of multi-protein complexes, localization or stability of the protein.
The region comprising residues 21–83 of greglin is a non-classical Kazal domain containing four disulfide bonds, of which three superposed on those of AEI, which belongs to the non-classical Kazal sub-group 1 . Compared with other Kazal members, greglin possesses a unique additional C-terminal region (residues 70–83) that folds back and is connected to the α-helix via a supplementary disulfide bond (Cys53–Cys76).
The overall fold of the Kazal domain of greglin is highly resistant to denaturation
Greglin (region 21–78) displays a higher thermostability than ovomucoid inhibitors. Our study shows that greglin retains complete inhibitory activity after 1 h at 95 °C, whereas the chicken ovomucoid inhibitor displays only residual activity (Fig. 4). Similarly, OMTKY3 was shown by circular dichroism to be unfolded at 90 °C, with a Tm of 58 °C . Several structural features may explain this protein thermostability. Factors contributing to protein stability include additional intramolecular interactions. Vogt et al. demonstrated that the thermostability of proteins is correlated with the number of hydrogen bonds . Moreover, disulfide bridges, another type of intermolecular interaction, are believed to stabilize proteins primarily through an entropic effect, by decreasing the entropy of the unfolded state of the protein . On the other hand, based on a comparative analysis of 20 complete genomes of thermophilic and mesophilic bacteria, Thompson and Eisenberg proposed the existence of a natural strategy for enhancing protein thermostability through truncations of exposed loop regions to lower the entropy of unfolding . Interestingly, all of these features are found in the greglin structure: (a) the length of the loop between Cys#1 and Cys#2 is shortened, (b) the size of secondary structural elements is enlarged, with greglin possessing the longest β-sheet, stabilized by 11 hydrogen bonds, and one of the longest α-helices among the Kazal family, and (c) the number of disulfide bonds is increased, which includes a fourth bridge that connects the C-terminus extension to the α-helix.
A possible role for the C-terminal extension (70–78) of greglin in inhibitory selectivity
Another feature of greglin is its preference for subtilisin (S8 family) over proteases of the S1 family, whereas OMTKY3 displays a broad inhibition range, with similar inhibitory constants (10−11–10−12 m) against proteases from the S1 and S8 families . The largest difference between greglin and OMTKY3 occurs for α-chymotrypsin, with Ki values of 2.6 × 10−8 m and 5.5 × 10−12 m, respectively.
Although the P1 position is the predominant determinant for the specificity, its influence may be exaggerated, as illustrated by OMTKY3, which inhibits a panel of proteases to an identical degree. In greglin, the P1 residue is Leu27, identical to the P1 of OMTKY3, and thus does not explain the differences in the inhibitory constants. OMTKY3 is one of the best-studied protein inhibitors, particularly at the structural level. We took advantage of the wealth of crystal structures to explore the binding to α-chymotrypsin. A fine superposition of the four complexes subtilisin–greglin, subtilisin–OMTKY3 (PDB ID 1R0R), α-chymotrypsin–OMTKY3 (PDB ID 1CHO) and HLE–OMTKY3 (PBD ID 1PPF) was performed for the RSLs in order to obtain the smallest root mean square deviation from residues P3 to (Fig. S8). The RSLs of OMTKY3 from the three complexes are perfectly superposed, with maximal deviations of 0.33 Å between the Cα atoms. The extremities of three side chains are reoriented: Leu18 (P1), with a small shift of 1.25 Å at the CG atom, Glu19 (), with a shift of 3.25 Å at the CD atom, and Arg21 (), with a shift of 3.25 Å at the CZ atom. Greglin superposes particularly well to OMTKY3 in complex with subtilisin. Because the side chains at and are shorter in greglin than in OMTKY3, their possible reorientation is expected to occur without any steric clash. Therefore, the structure of greglin from P3 to is entirely compatible for interaction with chymotrypsin. The reason for its preference for subtilisin must reside in other regions of greglin. For example, in the pacifastin family, we demonstrated a role for the P10–P6 region of the inhibitors in modulation of the selectivity of inhibition [32, 33].
Despite the active sites comprising superposable catalytic triads, the proteases from the S1 and S8 families display completely different folds and different loops surrounding the entrance of the active site. In the S1 family, the active-site cleft is shaped by several insertion loops, such as the 30-loop, the 60-loop, the 140-loop (the so-called autolysis loop) and the 220-loop, which have been described as involved in the specificity [34, 35]. The binding of greglin to chymotrypsin was modelled using the crystal structures of three complexes: subtilisin–OMTKY3 , chymotrypsin–OMTKY3  and chymotrypsin–PMP-D2v (PDB ID 1GL0) . As shown in Fig. 5 (right), in the modelled complex, the C-terminal extension of greglin points toward the autolysis loop of chymotrypsin. The distance between the Cα atoms of residue 73 in greglin and residue 149 in chymotrypsin is <2.0 Å. For comparison, in the subtilisin complex (Fig. 5, left), the smallest Cα–Cα distance in this region is 6.74 Å, and involves residue 73 of greglin and residue 157 of subtilisin. Therefore, in the modelled greglin–chymotrypsin complex, a steric clash is predicted to occur in the absence of reorganization of the loops. The length and amino acid composition of the autolysis loop is highly variable; for example, it is one residue longer in chymotrypsin than in elastases. The autolysis loop may adopt various conformations  to modulate the specificity of the enzyme, as demonstrated in the case of coagulation proteases . Thus, due to the flexibility of the autolysis loop, binding of greglin to proteases of the S1 family is ensured but is energetically less favourable, as illustrated by the moderate inhibition of not only chymotrypsin but also elastase and cathepsin G.
Figure 5. Comparison of the interaction of greglin with proteases from the S8 (left) and S1 (right) families. Greglin is represented as in Fig. 1. Left: crystal structure of the complex between greglin and subtilisin (of the S8 family), represented as a grey surface. Right: an identical orientation showing the complex between greglin and chymotrypsin (taken from complex PDB ID 1GL0). Chymotrypsin is a member of the S1 family, and is shown in surface representation, coloured in light brown, with its autolysis loop shown in orange. The figures were prepared using PyMOL.
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In conclusion, this structural study of greglin using both X-ray crystallography and mass spectrometry resulted in a correction of the protein sequence, which allows definition of a new sub-family of Kazal inhibitors, and provides insight into the structure–function relationship in terms of thermostability and selectivity. However, further studies are necessary to understand the role of the N-terminal domain.