Dynamics and Function of sRNA/mRNAs Under the Scrutiny of Computational Simulation Methods.

Molecular dynamics simulations have proved extremely useful in investigating the functioning of proteins with atomic-scale resolution. Many applications to the study of RNA also exist, and their number increases by the day. However, implementing MD simulations for RNA molecules in solution faces challenges that the MD practitioner must be aware of for the appropriate use of this tool. In this chapter, we present the fundamentals of MD simulations, in general, and the peculiarities of RNA simulations, in particular. We discuss the strengths and limitations of the technique and provide examples of its application to elucidate small RNA's performance.

Expanding the repertoire of human tandem repeat RNA-binding proteins.

Protein regions consisting of arrays of tandem repeats are known to bind other molecular partners, including nucleic acid molecules. Although the interactions between repeat proteins and DNA are already widely explored, studies characterising tandem repeat RNA-binding proteins are lacking. We performed a large-scale analysis of human proteins devoted to expanding the knowledge about tandem repeat proteins experimentally reported as RNA-binding molecules. This work is timely because of the release of a full set of accurate structural models for the human proteome amenable to repeat detection using structural methods. The main goal of our analysis was to build a comprehensive set of human RNA-binding proteins that contain repeats at the sequence or structure level. Our results showed that the combination of sequence and structural methods finds significantly more tandem repeat proteins than either method alone. We identified 219 tandem repeat proteins that bind RNA molecules and characterised the overlap between repeat regions and RNA-binding regions as a first step towards assessing their functional relationship. We observed differences in the characteristics of repeat regions predicted by sequence-based or structure-based methods in terms of their sequence composition, their functions and their protein domains.

Recognition and Binding of RsmE to an AGGAC Motif of RsmZ: Insights from Molecular Dynamics Simulations.

CsrA/RsmE is a post-transcriptional regulator protein widely distributed in bacteria. It impedes the expression of target mRNAs by attaching their 5' untranslated region. The translation is restored by small, noncoding RNAs that sequester CsrA/RsmE acting as sponges. In both cases, the protein recognizes and attaches to specific AGGAX and AXGGAX motifs, where X refers to any nucleotide. RsmZ of Pseudomonas protegens is one of these small RNAs. The structures of some of its complexes with RsmE were disclosed a few years ago. We have used umbrella sampling simulations to force the unbinding of RsmE from the AGGAC motif located in the single-stranded region sited between stem loops 2 and 3 of RsmZ. The calculations unveiled the identity of the main residues and nucleotides involved in the process. They also showed that the region adopts a hairpin-like conformation during the initial stages of the binding. The ability to acquire this conformation requires that the region has a length of at least nine nucleotides. Besides, we performed standard molecular dynamics simulations of the isolated fragments, analyzed their typical conformations, and characterized their movements. This analysis revealed that the free molecules oscillate along specific collective coordinates that facilitate the initial stages of the binding. The results strongly suggest that the flexibility of the single-stranded region of RsmZ crucially affects the ability of its binding motif to catch RsmE.

Molecular Dynamics Simulations Unveil the Basis of the Sequential Binding of RsmE to the Noncoding RNA RsmZ.

CsrA/RsmE are dimeric proteins that bind to targeted mRNAs repressing translation. This mechanism modulates several metabolic pathways and allows bacteria to efficiently adjust their responses to environmental changes. In turn, small RNAs (sRNA) such as CsrB or RsmZ, restore translation by sequestering CsrA/RsmE dimers. Thus, these molecules act in tandem as a gene-expression regulatory system. Recently, a combined NMR-EPR approach solved the structure of part of RsmZ of Pseudomonas fluorescens, attached to three RsmE dimers. The study demonstrated that RsmE assembles onto RsmZ following a specific sequential order. The reasons underlying this peculiar behavior are still unclear. Here, we present a molecular dynamics analysis that explores the conformational diversity of RsmZ and RsmZ-RsmE complexes. The results reveal a clear pattern regarding the exposure of the alternative GGA binding motifs of RsmZ. This pattern is tuned by the attachment of RsmE dimers. Altogether, the observations provide a simple and convincing explanation for the order observed in the sequestration of RsmE dimers. Typical structures for RsmZ and RsmZ-RsmE complexes have been identified. Their characteristics concerning the exposure of the GGA sequences are presented and their most significant interactions are described.

Positively Charged Residues in the Head Domain of P2X4 Receptors Assist the Binding of ATP.

P2X receptors are a family of trimeric cationic channels located in the membrane of mammalian cells. They open in response to the binding of ATP. The differences between the closed and open structures have been described in detail for some members of the family. However, the order in which the conformational changes take place as ATP enters the binding cleft, and the residues involved in the intermediate stages, are still unknown. Here, we present the results of umbrella sampling simulations aimed to elucidate the sequence of conformational changes that occur during the reversible binding of ATP to the P2X4 receptor. The simulations also provided information about the interactions that develop in the course of the process. In particular, they revealed the existence of a metastable state which assists the binding. This state is stabilized by positively charged residues located in the head domain of the receptor. Based on these findings, we propose a novel mechanism for the capture of ATP by P2X4 receptors.

Charge Discrimination in P2X4 Receptors Occurs in Two Consecutive Stages.

P2X receptors are a group of trimeric cationic channels that are activated by adenosine 5'-triphosphate. They perform critical roles in the membranes of mammalian cells, and their improper functioning is associated with numerous diseases. Despite the vast amount of research devoted to them, several aspects of their operation are currently unclear, including the causes of their charge selectivity. We present the results of molecular dynamics simulation, which shed light on this issue for the case of P2X4 channels. We examined in detail the behavior of Na+ and Cl- ions inside the receptor. The examination reveals that charge discrimination occurs in two stages. First, cations bear precedence over anions to enter the extracellular vestibule. Then, cations at the extracellular vestibule are more likely to cross the pore than anions in an equivalent position. In this manner, a thorough but straightforward analysis of computational simulations suggests a stepwise mechanism, without a unique determinant factor.