Protein tolerance to random amino acid change

Abstract

Mutagenesis of protein-encoding sequences occurs ubiquitously; it enables evolution, accumulates during aging, and is associated with disease. Many biotechnological methods exploit random mutations to evolve novel proteins. To quantitate protein tolerance to random change, it is vital to understand the probability that a random amino acid replacement will lead to a protein's functional inactivation. We define this probability as the "x factor." Here, we develop a broadly applicable approach to calculate x factors and demonstrate this method using the human DNA repair enzyme 3-methyladenine DNA glycosylase (AAG). Three gene-wide mutagenesis libraries were created, each with 10(5) diversity and averaging 2.2, 4.6, and 6.2 random amino acid changes per mutant. After determining the percentage of functional mutants in each library using high-stringency selection (>19,000-fold), the x factor was found to be 34% +/- 6%. Remarkably, reanalysis of data from studies of diverse proteins reveals similar inactivation probabilities. To delineate the nature of tolerated amino acid substitutions, we sequenced 244 surviving AAG mutants. The 920 tolerated substitutions were characterized by substitutability index and mapped onto the AAG primary, secondary, and known tertiary structures. Evolutionarily conserved residues show low substitutability indices. In AAG, beta strands are on average less substitutable than alpha helices; and surface loops that are not involved in DNA binding are the most substitutable. Our results are relevant to such diverse topics as applied molecular evolution, the rate of introduction of deleterious alleles into genomes in evolutionary history, and organisms' tolerance of mutational burden.

Fig. 1.

Tolerated amino acid changes along the AAG primary sequence, shown with evolutionary conservation and secondary structures. Two hundred forty-four active AAG mutants were sequenced, and observed amino acid substitutions are shown above the wild-type sequence. Colored bars indicate general categories of amino acids. Numbers are read vertically and indicate residue position. Below the wild-type sequence, evolutionarily invariant residues are marked by black boxes and conserved residues by gray boxes. α helices (helix), β strands (arrows), and disordered regions (dashed lines) are indicated. Homologous sequences (from human, mouse, rat, Borrelia burgdorferi, Bacillus subtilis, Arabidopsis thaliana, and Mycobacterium tuberculosis) were identified with psi-blast (10), and secondary structure calling was performed with molecular operating environment (moe, CCG, Montreal, Canada).

Fig. 2.

Substitutability of AAG amino acid residues and structure. Individual residues' substitutability scores are indicated by their color in the spectrum, with red being the most substitutable and dark blue the least. (A) The DNA interacting face of AAG. The DNA-binding face and intercalating Tyr-162 (^*) are largely intolerant of substitution, whereas distant loops are generally tolerant of change. (B) Rotation of A by 180°, showing the opposite side of the AAG surface. (C and D) The substitutability of AAG resides shown by secondary and tertiary structure representation. Views are rotated 180° relative to each other. Residues near the active site of AAG, adjacent to the extrahelical and 1,N⁶-ethenoadenine DNA lesion, are generally intolerant of change. The β4 (165–171) strand is indicated by #. Arrows point toward α helices with solvent accessible faces that exhibit greater substitutability than their buried sides.