The N-terminal RNA recognition motif domain (RRM1) of polypyrimidine tract binding protein (PTB) forms an additional C-terminal helix α3, which docks to one edge of the β-sheet upon binding to a stem-loop RNA containing a UCUUU pentaloop. Importantly, α3 does not contact the RNA. The α3 helix therefore represents an allosteric means to regulate the conformation of adjacent domains in PTB upon binding structured RNAs. Here we investigate the process of dynamic adaptation by stem-loop RNA and RRM1 using NMR and MD in order to obtain mechanistic insights on how this allostery is achieved. Relaxation data and NMR structure determination of the free protein show that α3 is partially ordered and interacts with the domain transiently. Stem-loop RNA binding quenches fast time scale dynamics and α3 becomes ordered, however microsecond dynamics at the protein-RNA interface is observed. MD shows how RRM1 binding to the stem-loop RNA is coupled to the stabilization of the C-terminal helix and helps to transduce differences in RNA loop sequence into changes in α3 length and order. IRES assays of full length PTB and a mutant with altered dynamics in the α3 region show that this dynamic allostery influences PTB function in cultured HEK293T cells.

• MeSH
• Allosteric Regulation MeSH
• Nucleic Acid Conformation MeSH
• Humans MeSH
• RNA Recognition Motif MeSH
• Polypyrimidine Tract-Binding Protein * metabolism   chemistry   MeSH
• Protein Domains MeSH
• RNA * chemistry   metabolism   MeSH
• Protein Folding MeSH
• Molecular Dynamics Simulation MeSH
• Protein Binding * MeSH
• Binding Sites MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Polypyrimidine Tract-Binding Protein * MeSH
• RNA * MeSH

The RNA recognition motif (RRM) is the most common RNA binding domain across eukaryotic proteins. It is therefore of great value to engineer its specificity to target RNAs of arbitrary sequence. This was recently achieved for the RRM in Rbfox protein, where four mutations R118D, E147R, N151S, and E152T were designed to target the precursor to the oncogenic miRNA 21. Here, we used a variety of molecular dynamics-based approaches to predict specific interactions at the binding interface. Overall, we have run approximately 50 microseconds of enhanced sampling and plain molecular dynamics simulations on the engineered complex as well as on the wild-type Rbfox·pre-miRNA 20b from which the mutated systems were designed. Comparison with the available NMR data on the wild type molecules (protein, RNA, and their complex) served to establish the accuracy of the calculations. Free energy calculations suggest that further improvements in affinity and selectivity are achieved by the S151T replacement.

• MeSH
• Nucleic Acid Conformation MeSH
• Humans MeSH
• MicroRNAs chemistry   genetics   metabolism   MeSH
• Models, Molecular MeSH
• RNA Recognition Motif * genetics   MeSH
• Nuclear Magnetic Resonance, Biomolecular MeSH
• Protein Engineering MeSH
• RNA-Binding Proteins chemistry   genetics   metabolism   MeSH
• RNA chemistry   metabolism   MeSH
• Amino Acid Sequence MeSH
• Molecular Dynamics Simulation MeSH
• RNA Stability MeSH
• Protein Binding MeSH
• Binding Sites genetics   MeSH
• Computational Biology MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• MicroRNAs MeSH
• MIRN20b microRNA, human MeSH Browser
• MIRN21 microRNA, human MeSH Browser
• RNA-Binding Proteins MeSH
• RNA MeSH

With both catalytic and genetic functions, ribonucleic acid (RNA) is perhaps the most pluripotent chemical species in molecular biology, and its functions are intimately linked to its structure and dynamics. Computer simulations, and in particular atomistic molecular dynamics (MD), allow structural dynamics of biomolecular systems to be investigated with unprecedented temporal and spatial resolution. We here provide a comprehensive overview of the fast-developing field of MD simulations of RNA molecules. We begin with an in-depth, evaluatory coverage of the most fundamental methodological challenges that set the basis for the future development of the field, in particular, the current developments and inherent physical limitations of the atomistic force fields and the recent advances in a broad spectrum of enhanced sampling methods. We also survey the closely related field of coarse-grained modeling of RNA systems. After dealing with the methodological aspects, we provide an exhaustive overview of the available RNA simulation literature, ranging from studies of the smallest RNA oligonucleotides to investigations of the entire ribosome. Our review encompasses tetranucleotides, tetraloops, a number of small RNA motifs, A-helix RNA, kissing-loop complexes, the TAR RNA element, the decoding center and other important regions of the ribosome, as well as assorted others systems. Extended sections are devoted to RNA-ion interactions, ribozymes, riboswitches, and protein/RNA complexes. Our overview is written for as broad of an audience as possible, aiming to provide a much-needed interdisciplinary bridge between computation and experiment, together with a perspective on the future of the field.

• MeSH
• DNA chemistry   MeSH
• Catalysis MeSH
• Nucleic Acid Conformation * MeSH
• Computer Simulation MeSH
• RNA chemistry   MeSH
• Molecular Dynamics Simulation * MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• DNA MeSH
• RNA MeSH