Deep Mutational Scanning of NDM-1: Reverse Engineering Antibiotic Resistance to Decode the Protein Sequence and Function Relationship

How does protein sequence affect function? How can mutations far from an enzyme’s active site radically alter its activity? Indeed, the metallo-β-lactamase NDM-4 contains a single mutation (M154L) located outside of its active site that enables enhanced carbapenem hydrolysis compared to NDM-1¹. The indirect effect of this mutation on catalytic activity demonstrates that predictions based on crystal structures alone provide an incomplete understanding of enzymatic activity and highlights the need for the comprehensive study of the role of every residue. Understanding how sequence affects structure and function is important for protein engineering because it allows us to better predict the functional effects of making sequence changes. Here, we perform deep mutational scanning on NDM-1 to explore the relationship between sequence, structure, and function, and facilitate rational design in protein engineering.

A comprehensive codon mutagenesis library for NDM-1 containing all 5,400 variants (270 positions x 20 possible amino acids) was created with restriction-free cloning. The enrichment of each variant will be determined at a high, middle, and low functional threshold for three β-lactam antibiotics: ampicillin, meropenem, and cefotaxime. With enrichment as a proxy for protein fitness, we will gain insight into the functional effects of each mutation at every position within the protein.

We expect that the knowledge gained from the deep mutational scanning of NDM-1 will be useful for future protein engineering endeavours. By assessing the role of every residue in NDM-1, we intend to generate a predictive model that may be applied to improve the design of enzymes with similar structure. In line with previous deep mutational scanning studies², this work has the potential to yield valuable information that may improve our understanding of how protein sequence affects function.

References
1. Nordmann, P., et al. Antimicrobial agents and chemotherapy, 2012.
2. Fowler, D.M., et al. Nature Methods 2014.