A systematic approach for evaluating the role of surface-exposed loops in trypsin-like serine proteases applied to the 170 loop in coagulation factor VIIa

Abstract

Proteases play a major role in many vital physiological processes. Trypsin-like serine proteases (TLPs), in particular, are paramount in proteolytic cascade systems such as blood coagulation and complement activation. The structural topology of TLPs is highly conserved, with the trypsin fold comprising two β-barrels connected by a number of variable surface-exposed loops that provide a surprising capacity for functional diversity and substrate specificity. To expand our understanding of the roles these loops play in substrate and co-factor interactions, we employ a systematic methodology akin to the natural truncations and insertions observed through evolution of TLPs. The approach explores a larger deletion space than classical random or directed mutagenesis. Using FVIIa as a model system, deletions of 1-7 amino acids through the surface exposed 170 loop, a vital allosteric regulator, was introduced. All variants were extensively evaluated by established functional assays and computational loop modelling with Rosetta. The approach revealed detailed structural and functional insights recapitulation and expanding on the main findings in relation to 170 loop functions elucidated over several decades using more cumbersome crystallization and single deletion/mutation methodologies. The larger deletion space was key in capturing the most active variant, which unexpectedly had a six-amino acid truncation. This variant would have remained undiscovered if only 2-3 deletions were considered, supporting the usefulness of the methodology in general protease engineering approaches. Our findings shed further light on the complex role that surface-exposed loops play in TLP function and supports the important role of loop length in the regulation and fine-tunning of enzymatic function throughout evolution.

Figure 1

FVII Structural Features and the Evolution of Surface-Exposed Loops. (A) Crystal structure of FVIIa:sTF (PDB ID:1dan, grey ), shown in standard representation. (B) Alignment of serine proteases from various stages of blood coagulation compared with the ancestral protease trypsin, together with the surface-exposed loops of FVIIa from various species (see supplemental Fig. S1). Conserved residues are shown in red, non-conserved in grey and the loop extension region in black. Both numbering based on the human Factor VII sequence and on the consensus Chymotrypsin numbering is shown. (C) The different conformations of the 170 loop in FVIIa, FVIIa-Y_T (4z6a, wheat ) and trypsin (3ptn, blue ) with deletion scanned residues in red and an N-glycan attachment site. (D) Deletion strategy in FVIIa with scanned residues in red and start positions for each deletion window marked with grey. A total of 39 variants were generated by moving the deletion windows through the loop.

Figure 2

Heatmap of Functional FVIIa Variant Characterization. Soluble tissue factor (sTF) binding as K_d [nM], amidolytic activity as K_M [mM], k_cat [s⁻¹] and percentage k_cat/K_M compared to FVIIa, pABA inhibition as percentage K_i of FVIIa and Factor X activation as percentage of FVIIa. More active or stronger binding variants are shown in blue and less active or weaker binding variants in red. All data is shown as the mean of duplicate runs without sTF. Variants are sorted by loop length and deletion position (in red) with the start of each sliding window underlined and the resulting loop length listed. (right bold numbering), the number of deleted residues is show on the left.

Figure 3

Functional FVIIa Data as a Function of Loop Length Normalized to FVIIa-WT in the absence of sTF (top) or presence of 3 μM sTF (bottom). (A) Functional data for hydrolysis of S-2288, (B) inhibition of FVIIa variants by pABA and (C) Activation of factor X. Group 1 consist of variants no. 12–14 and 20–22 while group 2 contains of variants no. 36, 37 and 39. Data is shown as the mean (n = 2) with high-low error bars for S-2288 and pABA, with mean ± SD (n = 4) for FX activation. The grey shaded area represents one standard deviation for FVIIa-WT around the mean (n = 6) from experiments conducted on different days and different plate positions. Loop length is the remaining number of residues in the 170-loop s for a give variant after a deletion, starting from the wild-type length of 14.

Figure 4

α-helix Angle Determines the Effects of Loop Length on 170 Loop Flexibility. (A) Orientation of the TF-helix as defined by the angle between the bottom of the 170 loop and the outer left side of the TF-helix in FVIIa (PDB ID 1dan, grey) and FVIIa-Y_T (PDB ID 4z6a, wheat) Q313{c170A} to P321{c170I} was rebuild using the Rosetta NGK protocol. (B) Average RMSF for all atoms rebuild in Rosetta with remaining loop lengths colour coded as red (13), green (12), blue (11), orange (10), grey (9), purple (8) and black (7). (C) Group 1 and group 2 FVIIa variants build using Rosetta Generalized Kinematic Closure (see supplemental Fig. S3 and S5 for all 39 variants). The root-mean-square fluctuation (RMSF) is defined around the shown geometric mean (N = 250) with low values shown in blue and high values in red.

Figure 5

Variants with an Intermediate Loop Length Show TF-Induced-Like Interactions. (A) The 170 loop region of FVIIa (PDB ID, 1dan) with the hydrogen bond between R315{c170C} and G372{c223} shown in red, together with the C310{c168}-C329{c182} disulphide-bridge in yellow. (B) The mean distance between the residue corresponding to the R315{c170C} backbone nitrogen and G372{c223} oxygen plotted against the standard deviation of the bond length for the FVIIa-WT (left panel) or FVIIa-Y_T backbone (right panel). Bond lengths of 2.5–3.8 Å are marked in grey for loop conformations with possible stable hydrogen bonds including variants 12–13 and 20–22. (C) Overview of group 1 variants with high activity including 10 modelled structures with the lowest Rosetta Energy (see supplemental Fig. S4) with the average hydrogen-bond distance shown in red. Frequency histograms for variants with distances in the 2.5–4.0 Å range showing a favourable distribution for loop lengths 11–12. The corresponding S-2288 activity relative to FVIIa in the right top-corner and deleted amino acids are coloured in red on the shown sequence.

Figure 6

Shorter Loop Lengths Allow for Trypsin-like Interactions with the Protease Domain. (A) Overview of trypsin-like interaction in a FVIIa-trypsin variant (PDB ID: 4z6a), with Y314 (172c) inserting into the protease domain, stabilizing Q366{c217} and F374{c225} by polar contacts (red) as observed in wild-type trypsin. (B) 10 models with lowest RMSD/REU of the most active variant (no 36.) from group 2, reconstructed using the NGK protocol. D319{c170G} makes polar contacts (red) with the backbone nitrogen of F374{c225} and sidechain of H373{c224} to stabilize the S1-pocket. A frequency histogram of the polar contacts between D319{c170G}-H373{c224} (grey) or D319-F374_N (red) highlights the favourability of the interaction (n = 250) with S-2288 activity relative to FVIIa in the right top-corner and deleted amino acids are coloured in red on the shown sequence. (C) Overview of variants 37–39 with 3 groups of 5 modelled structures selected from RMSD groupings showing the lowest REU (see supplemental Fig. S6). Average hydrogen-bond distance is shown in red when the distance is < 3.5 Å. Frequency histograms (n = 250) of the polar contacts between Q313{c170A} -F374_N{c225} (red) or Q313{c170A}-F374_O{c225} (purple).