Restriction of S-adenosylmethionine conformational freedom by knotted protein binding sites

Abstract

S-adenosylmethionine (SAM) is one of the most important enzyme substrates. It is vital for the function of various proteins, including large group of methyltransferases (MTs). Intriguingly, some bacterial and eukaryotic MTs, while catalysing the same reaction, possess significantly different topologies, with the former being a knotted one. Here, we conducted a comprehensive analysis of SAM conformational space and factors that affect its vastness. We investigated SAM in two forms: free in water (via NMR studies and explicit solvent simulations) and bound to proteins (based on all data available in the PDB). We identified structural descriptors -angles which show the major differences in SAM conformation between unknotted and knotted methyltransferases. Moreover, we report that this is caused mainly by a characteristic for knotted MTs tight binding site formed by the knot and the presence of adenine-binding loop. Additionally, we elucidate conformational restrictions imposed on SAM molecules by other protein groups in comparison to conformational space in water. Author summaryThe topology of a folded polypeptide chain has great impact on the resulting protein function and its interaction with ligands. Interestingly, topological constraints appear to affect binding of one of the most ubiquitous substrates in the cell, S-adenosylmethionine (SAM), to its target proteins. Here, we demonstrate how binding sites of specific proteins restrict SAM conformational freedom in comparison to its unbound state, with a special interest in proteins with non-trivial topology, including an exciting group of knotted methyltransferases. Using a vast array of computational methods combined with NMR experiments, we identify key structural features of knotted methyltransferases that impose unorthodox SAM conformations. We compare them with the characteristics of standard, unknotted SAM binding proteins. These results are significant for understanding differences between analogous, yet topologically different enzymes, as well as for future rational drug design. October 7, 2019 1/22 1 S-adenosylmethionine (SAM or AdoMet) is an ubiquitous molecule and the second most 2 widely used enzyme substrate after ATP [1]. It is utilized in many different chemical 3 reactions, including transfer of the methyl group. It acts as methyl donor for a variety 4 of methyltransferases (MTs), involved in methylation of small molecules [2], proteins [3], 5 DNA [4], and RNA [5]. Interestingly, SAM binds not only to proteins but also to RNA -6 it is a substrate for riboswitches [6]. 7The diversity of molecules capable of binding SAM is great not only in a variety of 8 performed functions, but also in their structure and topology. In the case of 9 SAM-dependent methyltransferases, the proteins are divided into five classes, each with 10 a different fold, including a knotted one [7]. The set of knotted methyltransferases 11 (MTs) is the largest group of all known knotted proteins, which structures comprise now 12 about 2% of PDB's deposits. Interestingly, all of t...

Mg2+-Dependent Methyl Transfer by a Knotted Protein: A Molecular Dynamics Simulation and Quantum Mechanics Study

“… 7 − 10 While the m 1 G37 methylation of tRNA is strictly conserved in all three domains of life, 7 , 11 it is catalyzed by TrmD in bacteria, but by Trm5 in Archaea and Eukarya. 12 In contrast to TrmD that requires Mg 2+ for catalysis, Trm5 needs no metal ions, a key distinction that separates the two enzymes, which also differ in sequence, 13 structure, 14 − 16 topology, 17 SAM binding, 18 , 19 and the mechanism of recognition of tRNA and the target base. 20 , 21 These fundamental differences, together with the essentiality of the m 1 G37-tRNA product for growth, have placed TrmD as a leading antimicrobial target.…”

“…This reaction produces m 1 G37-tRNA, which maintains protein synthesis reading frame and thus is essential for life. − While the m 1 G37 methylation of tRNA is strictly conserved in all three domains of life, , it is catalyzed by TrmD in bacteria, but by Trm5 in Archaea and Eukarya . In contrast to TrmD that requires Mg 2+ for catalysis, Trm5 needs no metal ions, a key distinction that separates the two enzymes, which also differ in sequence, structure, − topology, SAM binding, , and the mechanism of recognition of tRNA and the target base. , These fundamental differences, together with the essentiality of the m 1 G37-tRNA product for growth, have placed TrmD as a leading antimicrobial target . Additionally, the Mg 2+ -requirement of TrmD has been recently implicated in regulating the expression of the metal ion transporter gene mgtA, suggesting a broader impact in biology.…”