Functional significance of evolving protein sequence in dihydrofolate reductase from bacteria to humans

Abstract

With the rapidly growing wealth of genomic data, experimental inquiries on the functional significance of important divergence sites in protein evolution are becoming more accessible. Here we trace the evolution of dihydrofolate reductase (DHFR) and identify multiple key divergence sites among 233 species between humans and bacteria. We connect these sites, experimentally and computationally, to changes in the enzyme's binding properties and catalytic efficiency. One of the identified evolutionarily important sites is the N23PP modification (∼mid-Devonian, 415-385 Mya), which alters the conformational states of the active site loop in Escherichia coli dihydrofolate reductase and negatively impacts catalysis. This enzyme activity was restored with the inclusion of an evolutionarily significant lid domain (G51PEKN in E. coli enzyme; ∼2.4 Gya). Guided by this evolutionary genomic analysis, we generated a human-like E. coli dihydrofolate reductase variant through three simple mutations despite only 26% sequence identity between native human and E. coli DHFRs. Molecular dynamics simulations indicate that the overall conformational motions of the protein within a common scaffold are retained throughout evolution, although subtle changes to the equilibrium conformational sampling altered the free energy barrier of the enzymatic reaction in some cases. The data presented here provide a glimpse into the evolutionary trajectory of functional DHFR through its protein sequence space that lead to the diverged binding and catalytic properties of the E. coli and human enzymes.phylogenetic | EVB

Preservation of Protein Dynamics in Dihydrofolate Reductase Evolution

“…These calculations also indicated a restoration of narrower dynamics distribution for the N23PP/G51PEKN double mutant to a level similar to that of the WT (Fig. 3) (24). Yet, all of the rates and KIEs (e.g.…”

“…All synthesized materials were purified and stored as described previously (42). N23PP and N23PP/ G51PEKN ecDHFR were expressed and purified as described previously (3,24).…”

“…As shown in Table 2, the kinetic parameters of N23PP were restored to that of the WT through insertion of G51PEKN (24). To test the effects of the ecDHFR mutants on the nature of the catalyzed hydride transfer, the KIEs on the second-order rate constant (k cat /K m ) for N23PP and N23PP/ G51PEKN were measured for both H/T and D/T KIEs, and the resulting data were used to calculate the intrinsic KIEs.…”

“…Analysis of DHFR sequences from 233 species ranging from Escherichia coli to humans identified phylogenetically coherent events (24). These evolutionarily significant divergence sites include a polyproline sequence (PWPP) in the Met-20 loop region of the enzyme (position Asn-23 in ecDHFR; brown sphere in Fig.…”

“…Importantly, QM/MM simulations (24) indicated that on the fs-ns time scale the N23PP ecDHFR has a much broader range of dynamics (i.e. less "rigid") when going from the reactant state to the transition state than the WT.…”