Anti-Correlation between the Dynamics of the Active Site Loop and C-Terminal Tail in Relation to the Homodimer Asymmetry of the Mouse Erythroid 5-Aminolevulinate Synthase

Abstract

Biosynthesis of heme represents a complex process that involves multiple stages controlled by different enzymes. The first of these proteins is a pyridoxal 5′-phosphate (PLP)-dependent homodimeric enzyme, 5-aminolevulinate synthase (ALAS), that catalyzes the rate-limiting step in heme biosynthesis, the condensation of glycine with succinyl-CoA. Genetic mutations in human erythroid-specific ALAS (ALAS2) are associated with two inherited blood disorders, X-linked sideroblastic anemia (XLSA) and X-linked protoporphyria (XLPP). XLSA is caused by diminished ALAS2 activity leading to decreased ALA and heme syntheses and ultimately ineffective erythropoiesis, whereas XLPP results from “gain-of-function” ALAS2 mutations and consequent overproduction of protoporphyrin IX and increase in Zn2+-protoporphyrin levels. All XLPP-linked mutations affect the intrinsically disordered C-terminal tail of ALAS2. Our earlier molecular dynamics (MD) simulation-based analysis showed that the activity of ALAS2 could be regulated by the conformational flexibility of the active site loop whose structural features and dynamics could be changed due to mutations. We also revealed that the dynamic behavior of the two protomers of the ALAS2 dimer differed. However, how the structural dynamics of ALAS2 active site loop and C-terminal tail dynamics are related to each other and contribute to the homodimer asymmetry remained unanswered questions. In this study, we used bioinformatics and computational biology tools to evaluate the role(s) of the C-terminal tail dynamics in the structure and conformational dynamics of the murine ALAS2 homodimer active site loop. To assess the structural correlation between these two regions, we analyzed their structural displacements and determined their degree of correlation. Here, we report that the dynamics of ALAS2 active site loop is anti-correlated with the dynamics of the C-terminal tail and that this anti-correlation can represent a molecular basis for the functional and dynamic asymmetry of the ALAS2 homodimer.

Water-Triggered, Irreversible Conformational Change of SARS-CoV-2 Main Protease on Passing from the Solid State to Aqueous Solution

“…Although most X-ray studies on SC-2 M pro show an almost perfect symmetry of the homodimer, indirect evidence suggests that only one of the two subunits is catalytically active in each of the subunits, as in a variety of other homodimeric proteins . (i) In the almost identical homodimeric protein from SARS-CoV (SC M pro , 96% sequence identity with SC-2 M pro ), one subunit is inactive because of distortions of the active site; − molecular dynamics (MD) provided insight on this asymmetry, similarly to what has been done for other homodimeric proteins. , (ii) Different analyses of a 0.1-ms-long MD simulation, recently performed by D. E. Shaw’s research group (DESRES), of SC-2 M pro in aqueous solution suggested that each of the two subunits visits a different set of configurations − after starting from a symmetric X-ray structure (PDB 6Y84 ). This hints at a role of packing forces in the conformation of the protein .…”

An Extended C-Terminus, the Possible Culprit for Differential Regulation of 5-Aminolevulinate Synthase Isoforms

“…Yet, the CXXC motif is conserved in ALAS1, and if it has a redox switching function then some degree of conformational perturbation presumably occurs to form an autoinhibited conformation or to alter the dynamics about the active site loop, which controls the catalytic rate. This latter possibility is attractive as it would be consistent with the anti-correlation between the active site loop and C-terminal extension of ALAS2 during molecular dynamics simulations (Na et al, 2018). Additionally, the shielding of the otherwise solvent exposed HRMs 4 and 5 by the ALAS1 C-terminal extension suggests an alternative conformation that would allow heme access to feedback inhibit the enzyme.…”

The yeast ALA synthase C‐terminus positively controls enzyme structure and function

“…Paired with the differences in PLP occupancy at the two active sites, the differences in disorder of the Hem1 ΔCT subunits at the β-arm and GR point toward structural asymmetry that extends past cofactor binding and instead could be an inherent property of Hem1 that is controlled by the C-terminal extension. This asymmetry was also alluded to in bacterial and vertebrate ALAS enzymes (Bailey et al, 2020;Na et al, 2018).…”