A unified genetic, computational and experimental framework identifies functionally relevant residues of the homing endonuclease I-BmoI

Abstract

Insight into protein structure and function is best obtained through a synthesis of experimental, structural and bioinformatic data. Here, we outline a framework that we call MUSE (mutual information, unigenic evolution and structure-guided elucidation), which facilitated the identification of previously unknown residues that are relevant for function of the GIY-YIG homing endonuclease I-BmoI. Our approach synthesizes three types of data: mutual information analyses that identify co-evolving residues within the GIY-YIG catalytic domain; a unigenic evolution strategy that identifies hyper- and hypo-mutable residues of I-BmoI; and interpretation of the unigenic and co-evolution data using a homology model. In particular, we identify novel positions within the GIY-YIG domain as functionally important. Proof-of-principle experiments implicate the non-conserved I71 as functionally relevant, with an I71N mutant accumulating a nicked cleavage intermediate. Moreover, many additional positions within the catalytic, linker and C-terminal domains of I-BmoI were implicated as important for function. Our results represent a platform on which to pursue future studies of I-BmoI and other GIY-YIG-containing proteins, and demonstrate that MUSE can successfully identify novel functionally critical residues that would be ignored in a traditional structure-function analysis within an extensively studied small domain of approximately 90 amino acids.

Figure 1.

I-BmoI is a modular GIY-YIG homing endonuclease. (A) Schematic representation of I-BmoI interactions with intronless thyA substrate based on biochemical data (27,32). Top- and bottom-strand nicking sites are shown as open and filled triangles, respectively, and the critical −2 GC base pair is shown in enlarged, bold-type font. The intron insertion site is indicated by a vertical line, with exon 1 sequence upstream (−) and exon 2 sequence downstream (+). (B) Homology model of the I-BmoI catalytic domain (residues 1–88). Highlighted are four highly conserved residues in GIY-YIG alignments that are critical for function, and secondary structure elements of the domain. Subsequent illustrations of the catalytic domain will be shown from this view (front) or a 180-degree rotation (back). (C) Surface representation of a front view of the I-BmoI homology model highlighting the putative catalytic cleft. The side chains of Y17, R27, E74 and N87 are surface exposed, lie along the base of the cleft, and are situated in close proximity to one another. Patches of charge are shown in colour, blue being positive, red being negative and green being hydrophobic.

Figure 2.

Alignment of the GIY-YIG domain. (A) Multiple sequence alignment of 146 sequences represented as a sequence logo (50). Positions are numbered according to the I-BmoI sequence that is shown below the alignment (conserved functionally critical residues are shown in green). Predicted secondary structure elements of the I-BmoI GIY-YIG domain are indicated on the sequence by shading, and motif assignments are identified on the alignment with shaded boxes. (B) Front (left) and rear (right) views of the homology model with the degree of conservation mapped onto the structure. Conserved positions are shown in dark green, with the side chains of highly conserved residues indicated. Variable positions are shown in white.

Figure 3.

Co-evolving residues of the I-BmoI catalytic domain. Position of four sets of co-evolving residues mapped onto the I-BmoI homology model, colour-coded by co-evolving residues (yellow, L35, H40, N46, F49; red S20, I71; blue K7, T9, F16, E60; orange K51, H52). Front (left) and rear (right) views are shown, with functionally critical residues highlighted in light green.

Figure 4.

The two-plasmid genetic selection. (A) Schematic of the expression plasmid (pExp) and toxic (reporter) plasmid (pTox). (B) Verification of the genetic selection using variants of pExp and pTox. Survival rates are expressed as the ratio of colonies on chloramphenicol + arabinose plates to colonies on chloramphenicol only plates. WT I-BmoI, pExp expressing WT I-BmoI; R27A I-BmoI, pExp expressing an inactive R27A I-BmoI; randomized I-BmoI, library of I-BmoI variants; p11-lacY-wtx1, parental pTox without I-BmoI target site; ND, not determined.

Figure 5.

Unigenic evolution analysis of I-BmoI. Shown is a summary of the mutations found in the 87 selected clones and the EoS value for each position. Non-synonymous substitutions found at each position are indicated beneath the corresponding I-BmoI sequence over the entire length of the protein (multiple independent occurrences of the same mutation are shown in subscript). Shown above each line of sequence is a graph of the evidence of selection (EoS) at each amino-acid position of I-BmoI. Regions of modelled or predicted secondary structure are indicated by grey rectangles, and bold residues indicate the GIY and YIG motifs, functionally critical residues, or residues that are identical between I-BmoI and I-TevI in the linker domain.

Figure 6.

Mapping positions that tolerate non-synonymous substitutions and EoS data onto the I-BmoI homology model. For the left of each panel, amino-acid positions that tolerate non-synonymous substitutions are shown in red and positions where no change was found are in white. On the right of each panel, the EoS score is shown as gradient. Positions with high EoS values are blue, and low EoS values are white. (A) Ribbon representations of a front view of the catalytic domain, with functionally critical residues identified by the present and previous studies indicated. (B) Surface representation of a front view of the I-BmoI catalytic domain, with the putative catalytic cleft bounded by white dashed lines. (C) Surface representation of a rear view of the catalytic domain.

Figure 7.

Cleavage activity of WT I-BmoI and site-directed mutants. (A) Shown are representative cleavage assays using 2-fold serial dilutions of the WT and mutant I-BmoI proteins, from 700 nM on the right to 1 nM on the left. The second lane from the left (-) of each gel image is unreacted substrate. (B) For the R27A, H31A and N42D mutants, only the three highest protein concentrations were tested. Substrate linearized by EcoRI is shown in the third lane from the left. Circular (C), linear (L) and nicked (N) plasmid forms are indicated to the right of each gel image.

Figure 8.

Nicking assays with WT and mutant proteins. Shown are representative agarose gels of time-course assays, with time points in seconds indicated above the gels. The second lane of each gel from the left contains unreacted substrate (−). Circular (C), linear (L) and nicked (N) plasmid forms are indicated to the right of each gel. Beside each gel image is a graphical representation of the disappearance of substrate and appearance of products formed over time. Data points from three independent experiments are plotted, with a continuous line drawn through the average.

Figure 9.

Summary of functionally relevant residues identified by MUSE. Shown are ribbon (left) and surface (right) representations of the I-BmoI catalytic domain with the residues identified by MUSE (S20, H31, N42, I67 and I71) highlighted in green, and previously identified functionally critical residues (Y17, R27, E74 and N87) shown in black. The catalytic cleft is highlighted as in Figure 1C.