Molecular insights into binding dynamics of tandem RNA recognition motifs (tRRMs) of human antigen R (HuR) with mRNA and the effect of point mutations in impaired HuR-mRNA recognition.

Human antigen R (HuR) is a key regulatory protein with prominent roles in RNA metabolism and post-transcriptional gene regulation. Many studies have shown the involvement of HuR in plethora of human diseases, which are often manifestations of impaired HuR-RNA interactions. However, the inherent complexities of highly flexible protein-RNA interactions have limited our understanding of the structural basis of HuR-RNA recognition. In this study, we dissect the underlying molecular mechanism of interaction between N-terminal tandem RNA-recognition motifs (tRRMs) of HuR and mRNA using molecular dynamics simulation. We have also explored the effect of point mutations (T90A, R97A and R136A) of three reported critical residues in HuR-mRNA binding specificity. Our findings show that N-terminal tRRMs exhibit conformational stability upon RNA binding. We further show that R136A and R97A mutants significantly lose their binding affinity owing to the loss of critical interactions with mRNA. This may be attributed to the larger domain rearrangements in the mutant complexes, especially the ß2ß3 loops in both the tRRMs, leading to unfavourable conformations and loss of binding affinity. We have identified critical binding residues in tRRMs of HuR, contributing favourable binding energy in mRNA recognition. This study contributes significantly to understand the molecular mechanism of RNA recognition by tandem RRMs and provides a platform to modulate binding affinities through mutations. This may further guide in future structure-based drug-therapies targeting impaired HuR-RNA interactions.Communicated by Ramaswamy H. Sarma.

Impaired nuclear transport induced by juvenile ALS causing P525L mutation in NLS domain of FUS: A molecular mechanistic study.

Amyotrophic lateral sclerosis (ALS) and fronto-temporal lobar degeneration (FTLD) are progressive neurological disorders affecting motor neurons. Cellular aggregates of fused in sarcoma (FUS) protein are found in cytoplasm of ALS and FTLD patients. Nuclear localisation signal (NLS) domain of FUS binds to Karyopherin ß2 (Kapß2), which drives nuclear transport of FUS from cytoplasm. Several pathogenic mutations are reported in FUS NLS, which are associated with its impaired nuclear transport and cytoplasmic mis-localisation. P525L mutation in NLS is most commonly found in cases of juvenile ALS (jALS), which affects individuals below 25 years of age. jALS progresses aggressively causing death within a year of its onset. This study elucidates the molecular mechanism behind jALS-causing P525L mutation hindering nuclear transport of FUS. We perform multiple molecular dynamics simulations in aqueous and hydrophobic solvent to understand the effect of the mutation at molecular level. Dynamics of Kapß2-FUS complex is better captured in hydrophobic solvent compared to aqueous solvent. P525 and Y526 (PY-motif) of NLS exhibit fine-tuned stereochemical arrangement, which is essential for optimum Kapß2 binding. P525L causes loss of several native contacts at interface leading to weaker binding, which promotes self-aggregation of FUS in cytoplasm. Native complex samples closed conformation, while mutant complex exhibits open conformation exposing hydrophilic residues of Kapß2 to hydrophobic solvent. Mutant complex also fails to exhibit spring-like motion essential for its transport through nuclear pore complex. This study provides a mechanistic insight of binding affinity between NLS and Kapß2 that inhibits self-aggregation of FUS preventing the disease condition.

Unusual RNA binding of FUS RRM studied by molecular dynamics simulation and enhanced sampling method.

Amyotrophic lateral sclerosis (ALS) and frontotemporal lobe degeneration (FTLD) are two inter-related intractable diseases of motor neuron degeneration. Fused in sarcoma (FUS) is found in cytoplasmic accumulation of ALS and FTLD patients, which readily link the protein with the diseases. The RNA recognition motif (RRM) of FUS has the canonical α-ß folds along with an unusual lysine-rich loop (KK-loop) between α1 and ß2. This KK-loop is highly conserved among FET family proteins. Another contrasting feature of FUS RRM is the absence of critical binding residues, which are otherwise highly conserved in canonical RRMs. These residues in FUS RRM are Thr286, Glu336, Thr338, and Ser367, which are substitutions of lysine, phenylalanine, phenylalanine, and lysine, respectively, in other RRMs. Considering the importance of FUS in RNA regulation and metabolism, and its implication in ALS and FTLD, it is important to elucidate the underlying molecular mechanism of RNA recognition. In this study, we have performed molecular dynamics simulation with enhanced sampling to understand the conformational dynamics of noncanonical FUS RRM and its binding with RNA. We studied two sets of mutations: one with alanine mutation of KK-loop and another with KK-loop mutations along with critical binding residues mutated back to their canonical form. We find that concerted movement of KK-loop and loop between ß2 and ß3 facilitates the folding of the partner RNA, indicating an induced-fit mechanism of RNA binding. Flexibility of the RRM is highly restricted upon mutating the lysine residues of the KK-loop, resulting in weaker binding with the RNA. Our results also suggest that absence of the canonical residues in FUS RRM along with the KK-loop is equally important in regulating its binding dynamics. This study provides a significant structural insight into the binding of FUS RRM with its cognate RNA, which may further help in designing potential drugs targeting noncanonical RNA recognition.

DSS1 allosterically regulates the conformation of the tower domain of BRCA2 that has dsDNA binding specificity for homologous recombination.

DSS1 is an evolutionary conserved, small intrinsically disordered protein that regulates various cellular functions. Although several studies have elucidated the role of DSS1 in stabilizing BRCA2 and its importance in homologous recombination repair (HRR), yet the structural mechanism behind the stability and HRR remains elusive. In this study, using molecular dynamics simulation we show that DSS1 stabilizes linearly arranged DNA/DSS1 binding domains of BRCA2 with many native contacts. These contacts are absent in the complexes with two missense DSS1 mutants associated with germline breast cancer and somatic mouth carcinoma. Most importantly, our protein energy-based network models show DSS1 allosterically regulates the conformation of the distant tower domain of BRCA2 that has dsDNA binding specificity for HRR. We further postulate that the unique conformation of the tower domain with kinked-helices might be responsible for DNA strand invasion and initiation of HRR. Induced conformation of the tower domain by the kinked-helices is absent in the unbound BRCA2, as well as in the two mutant DSS1-BRCA2 complexes. This suggests that DSS1 allosterically regulates the tower domain conformations of BRCA2 that affects dsDNA binding, essential for HRR. Our results add a new dimension to the function of DSS1 and its role in regulating HRR.