Conserved amino acid networks modulate discrete functional properties in an enzyme superfamily

Abstract

In this work, we applied the sequence-based statistical coupling analysis approach to characterize conserved amino acid networks important for biochemical function in the pancreatic-type ribonuclease (ptRNase) superfamily. This superfamily-wide analysis indicates a decomposition of the RNase tertiary structure into spatially distributed yet physically connected networks of co-evolving amino acids, termed sectors. Comparison of this statistics-based description with new NMR experiments data shows that discrete amino acid networks, termed sectors, control the tuning of distinct functional properties in different enzyme homologs. Further, experimental characterization of evolutionarily distant sequences reveals that sequence variation at sector positions can distinguish homologs with a conserved dynamic pattern and optimal catalytic activity from those with altered dynamics and diminished catalytic activities. Taken together, these results provide important insights into the mechanistic design of the ptRNase superfamily, and presents a structural basis for evolutionary tuning of function in functionally diverse enzyme homologs.

Engineered control of enzyme structural dynamics and function

“…This method provided atomic‐level insight into how distal amino acid substitutions, like G121V and S148A, can propagate their effects throughout the enzyme. Similar types of network analyses have contributed to our understanding in many other enzyme systems …”

“…Similar types of network analyses have contributed to our understanding in many other enzyme systems. [101][102][103][104][105][106][107] These long-range networks may also offer means through which enzymes communicate in multi-enzyme complexes. 97 For example, we used NMR chemical shift covariance analysis 108,109 among a series of sitedirected protein variants to identify amino acid interaction networks in the alpha subunit of E. coli tryptophan synthase (aTS).…”

Ligand-Induced Variations in Structural and Dynamical Properties Within an Enzyme Superfamily

“…The rate-limiting step was previously shown to correspond to a conformational change in a distal loop that is associated with the product release step in RNase A (Watt et al, 2011 ; Gagné and Doucet, 2013 ). The functional role of conformational exchange in product release was previously shown to rely on the movement of distal loop regions in RNase A, a hypothesis that we further extended to include functional RNase homologs sharing a conserved structural fold (Cole and Loria, 2002 ; Watt et al, 2007 ; Doucet et al, 2009 , 2011 ; Gagné et al, 2012 ; Gagné and Doucet, 2013 ; Narayanan et al, 2017 , 2018 ). Mutations of residues in these loop regions were shown to result in reduced rate constants for product release and lower substrate affinity, highlighting the role of these long-range motions in this enzyme (Gagné and Doucet, 2013 ).…”

“…In addition to their common ribonucleolytic function, the canonical RNases, henceforth referred to as subtypes , have evolved to perform other biological functions such as host defense, immunosuppressivity, angiogenesis, and anti-pathogenic activity, among others (Sorrentino, 2010 ). Further, the experimentally characterized human RNase subtypes display a wide range of substrate specificities (Boix et al, 2013 ), catalytic activities (Sorrentino, 2010 ; Gagné and Doucet, 2013 ) and conformational fluctuations on the millisecond timescale (Narayanan et al, 2017 , 2018 ). Efforts to relate specific conformational exchange events with ribonucleolytic function in this enzyme family is thus limited by the broader and often RNA-independent biological functions of many homologous RNase superfamily members.…”

“…Coevolving amino acid residues that are proximal to each other in the three-dimensional structure may control the biological properties of proteins (Halabi et al, 2009 ). In a recent study, we identified networks of coevolving amino acids that control distinct aspects of biochemical functions in the pancreatic RNase superfamily by combining the sequence-based statistical analysis with experimental observations (Narayanan et al, 2017 ). Our results demonstrated that networks of residues within a larger functional sector are involved in fine-tuning the catalytic activity among the different RNase subtypes, thus dictating the functional diversity among these RNases.…”