Exploring the Evolutionary History of Kinetic Stability in the α-Lytic Protease Family

Abstract

In addition to encoding the tertiary fold and stability, the primary sequence of a protein encodes the folding trajectory and kinetic barriers that determine the speed of folding. How these kinetic barriers are encoded is not well understood. Here, we use evolutionary sequence variation in the α-lytic protease (αLP) protein family to probe the relationship between sequence and energy landscape. αLP has an unusual energy landscape: the native state of αLP is not the most thermodynamically favored conformation and, instead, remains folded due to a large kinetic barrier preventing unfolding. To fold, αLP utilizes an N-terminal pro region similar in size to the protease itself that functions as a folding catalyst. Once folded, the pro region is removed, and the native state does not unfold on a biologically relevant time scale. Without the pro region, αLP folds on the order of millennia. A phylogenetic search uncovers αLP homologs with a wide range of pro region sizes, including some with no pro region at all. In the resulting phylogenetic tree, these homologs cluster by pro region size. By studying homologs naturally lacking a pro region, we demonstrate they can be thermodynamically stable, fold much faster than αLP, yet retain the same fold as αLP. Key amino acids thought to contribute to αLP's extreme kinetic stability are lost in these homologs, supporting their role in kinetic stability. This study highlights how the entire energy landscape plays an important role in determining the evolutionary pressures on the protein sequence.

Figure 1.

A) Histogram of αLP homolog pro region sizes fit to a set of normal distributions (red dashed line), with the peaks represented as blue dots. Pro region ranges (defined as peak ± two standard deviations) are highlighted as No-pro (purple), ProS (blue), ProM (green), and ProL (red). B) αLP complexed with its pro region (PDB ID: 4PRO) and pro region truncations to represent presumed structures (based on the structure of the pro region of αLP) of ProM, ProS, and No-pro homologs, based on peak pro-region size values. αLP protease N-terminal domain is in black and C-terminal domain is in grey.

Figure 2.

A) αLP phylogenetic tree with clades collapsed and colored based on pro regions found within that clade: No-pro (purple), ProS (blue), ProM (green), and ProL (red). Each clade is annotated with the number of sequences of each pro region cluster/color. The raw log-likelihood ratio and SH support (in parentheses) are listed for nodes where the pro region size changes. B) Alignment of selected homologs. The sequences are colored based on pro region and protease domains. Active site residues (canonical Asp102, His57, and Ser195) are noted in red boxes. (Asterisk) indicates fully conserved residues, (colon) indicates strongly similar residues, and (period) indicated weakly similar residues.

Figure 3.

A) Normalized chemical denaturation melts monitored by tryptophan fluorescence of homologs N2 (circles), N3 (squares), and N4 (triangles). Fit lines represent a global fit of replicate melts with linked folded and unfolded baselines. B) ln(k_obs) vs denaturant concentration for unfolding kinetic traces of homologs N2 (circles), N3 (squares), and N4 (triangles). Linear fits of the data were used to extrapolate the unfolding rate in 0 M denaturant for each homolog.

Figure 4.

A) Superimposed ribbon diagram structures of N4 (purple) and αLP (PDB ID 4PRO; grey). Canonical catalytic triad residues are represented as atoms. Zoom inset of catalytic triad. B) N4 structure rendered by average residue B factor/residue (max 48.12, min 11.87). C) Three loops in N4 are longer than the respective loops in αLP. Overall random coil structure is 5% higher for N4 (21% vs. 16%). D) Sequence logo of Phe228 and co-varying residues for each pro region category and the individual residues of αLP and N4. Residue numbering is based on those previously used for αLP, which is based on homology to chymotrypsin.

Figure 5.

A) Contact map for αLP (UniProtKB - P00778) generated by EVcouplings (pro residues 25-199 and protease residues 200-397; numbering based on UniProtKB entry). Top intermolecular couplings are circled (yellow). B) αLP protease (grey) and pro region (red) structure with the top intermolecular couplings highlighted (yellow). C) Sequence logo of top couplings between the pro and protease region for ProL and No-pro homologs.