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Tuning the relative affinities for activating and repressing operators of a temporally regulated restriction-modification system.

ABSTRACT: Most type II restriction-modification (R-M) systems produce separate endonuclease (REase) and methyltransferase (MTase) proteins. After R-M genes enter a new cell, MTase activity must appear before REase or the host chromosome will be cleaved. Temporal control of these genes thus has life-or-death consequences. PvuII and some other R-M systems delay endonuclease expression by cotranscribing the REase gene with the upstream gene for an autogenous activator/repressor (C protein). C.PvuII was previously shown to have low levels early, but positive feedback later boosts transcription of the C and REase genes. The MTase is expressed without delay, and protects the host DNA. C.PvuII binds to two sites upstream of its gene: O(L), associated with activation, and O(R), associated with repression. Even when symmetry elements of each operator are made identical, C.PvuII binds preferentially to O(L). In this study, the intra-operator spacers are shown to modulate relative C.PvuII affinity. In light of a recently reported C.Esp1396I-DNA co-crystal structure, in vitro and in vivo effects of altering O(L) and O(R) spacers were determined. The results suggest that the GACTnnnAGTC consensus is the primary determinant of C.PvuII binding affinity, with intra-operator spacers playing a fine-tuning role that affects mobility of this R-M system.

SUBMITTER: Mruk I

PROVIDER: S-EPMC2647307 | biostudies-literature | 2009 Feb

REPOSITORIES: biostudies-literature

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Tuning the relative affinities for activating and repressing operators of a temporally regulated restriction-modification system.

Mruk Iwona I Blumenthal Robert M RM

Nucleic acids research 20090106 3

Most type II restriction-modification (R-M) systems produce separate endonuclease (REase) and methyltransferase (MTase) proteins. After R-M genes enter a new cell, MTase activity must appear before REase or the host chromosome will be cleaved. Temporal control of these genes thus has life-or-death consequences. PvuII and some other R-M systems delay endonuclease expression by cotranscribing the REase gene with the upstream gene for an autogenous activator/repressor (C protein). C.PvuII was previ ...[more]

PMID: 19126580

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Project description:A growing number of computational tools have been developed to accurately and rapidly predict the impact of amino acid mutations on protein-protein relative binding affinities. Such tools have many applications, for example, designing new drugs and studying evolutionary mechanisms. In the search for accuracy, many of these methods employ expensive yet rigorous molecular dynamics simulations. By contrast, non-rigorous methods use less exhaustive statistical mechanics, allowing for more efficient calculations. However, it is unclear if such methods retain enough accuracy to replace rigorous methods in binding affinity calculations. This trade-off between accuracy and computational expense makes it difficult to determine the best method for a particular system or study. Here, eight non-rigorous computational methods were assessed using eight antibody-antigen and eight non-antibody-antigen complexes for their ability to accurately predict relative binding affinities (ΔΔG) for 654 single mutations. In addition to assessing accuracy, we analyzed the CPU cost and performance for each method using a variety of physico-chemical structural features. This allowed us to posit scenarios in which each method may be best utilized. Most methods performed worse when applied to antibody-antigen complexes compared to non-antibody-antigen complexes. Rosetta-based JayZ and EasyE methods classified mutations as destabilizing (ΔΔG < -0.5 kcal/mol) with high (83-98%) accuracy and a relatively low computational cost for non-antibody-antigen complexes. Some of the most accurate results for antibody-antigen systems came from combining molecular dynamics with FoldX with a correlation coefficient (r) of 0.46, but this was also the most computationally expensive method. Overall, our results suggest these methods can be used to quickly and accurately predict stabilizing versus destabilizing mutations but are less accurate at predicting actual binding affinities. This study highlights the need for continued development of reliable, accessible, and reproducible methods for predicting binding affinities in antibody-antigen proteins and provides a recipe for using current methods.

Dataset Information

Tuning the relative affinities for activating and repressing operators of a temporally regulated restriction-modification system.

Publications

Tuning the relative affinities for activating and repressing operators of a temporally regulated restriction-modification system.

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