TABLE 1.
Summary of altered specificities and activities
Variants are grouped into categories (C-terminal loops, K200, central 4 loops, core mutations) dependent on the location and theorized role of the mutations transferred to the I-AniI scaffold. The base activity column indicates whether the variant was made with either of the activating F13Y or S111Y mutations. Quantitative activities, cleavage plots, and additional information on each variant are available under supplemental Table S2 and Figs. S4–S8.
| Variant | Mutations from I-AniI | Base activity | Tested positions | Effect on specificity and activity |
|---|---|---|---|---|
| C-terminal loops | ||||
| I-PnoIP | V153I, N157H, D159E, D160E, I164V | F13Y | +8, +9, +10 | Specificity pattern similar to I-AniI; activity increased for most substitutions |
| I-AchIP | V153I, K155N, L156T, N157K, D160T, inserted A after 160, D161T, I164A | F13Y | +7, +8, +9, +10 | Novel +7A specificity; loss of specificity at +8, +9, and +10 |
| I-TasIP | K155T, L156R, N157T, K158 deletion, D160H, D161S, Y162S, L163K | F13Y | +7, +8, +9, +10 | Shifts favoring +7A, +8G, and +9C |
| K200 | ||||
| I-NfiIP | I164V, K200N | F13Y | +3, +4 | Novel +3C specificity; similar specificity to I-AniI at other substitutions |
| I-PnoIP | K200R | F13Y | +3, +4, +5 | Increased cleavage of +3G and +4T |
| Central 4 loops | ||||
| I-GzeIP | R172K, T196Y, N197D | F13Y | +2, +3 | Specificity pattern similar to I-AniI; activity increased for most substitutions |
| I-AchIP | K60T, R61K, N62K, E63D, inserted G after 63, I64T, E65K, M66L | S111Y | −2 | Purines preferred over pyrimidines |
| I-VinIP loop | S57G, K60D, N62T, E63L, I64V, SGVVS insert after 64, E65K, A68Y, L69F, R70K, I71V | S111Y | −2 | −2C favored over WT −2T; matches −2C target site prediction |
| Core mutations | ||||
| I-VinIP without core | A68Y, R70K | WT | −6, −5 | Minimal activity observed on −6 and −5 targets for variant with interface mutations only; activity recovered with addition of core mutations L69F and I71V |
| I-VinIP with core | A68Y, L69F, R70K, I71V | WT | ||
| I-VinIP-S111Y without core | A68Y, R70K | S111Y | −6, −5 | Novel −6T specificity; enhanced cleavage activity due to core mutations |
| I-VinIP-S111Y with core | A68Y, L69F, R70K, I71V | S111Y | ||
| I-VinIP Loop | S57G, K60D, N62T, E63L, I64V, SGVVS insert after 64, E65K, A68Y, L69F, R70K, I71V | S111Y | −6, −5 | Activity increased further relative to I-VinIP-S111Y w/core |
| K24N/L28V/T29K | K24N, L28V, T29K | WT | −8 | L28V core mutation enhances activity; computationally predicted K24N and T29K highly specific for −8G; matches −8G target site prediction |
| K24N/T29K | K24N, T29K, lacking L233K | WT | ||
| I-PnoIP N-terminal transfera | Y18F, S20T, I21V, T29A, A68S | S111Y | −6, −5 | −5T substitution preferred over WT −5A; matches −5T target site prediction |
a Quantitative data and cleavage plots for this variant, included as an additional example of transferred mutations conferring specificity towards the putative homologue target site, are given under supplemental data.