A cytosine-patch sequence motif identified in the conserved region of lincRNA-p21 interacts with the KH3 domain of hnRNPK.

Long Intergenic Non-coding RNAs (lincRNAs) are the largest class of long non-coding RNAs in eukaryotes, originating from the genome's intergenic regions. A ∼4 â€‹kb long lincRNA-p21 is derived from a transcription unit next to the p21/Cdkn1a gene locus. LincRNA-p21 plays regulatory roles in p53-dependent transcriptional and translational repression through its physical association with proteins such as hnRNPK and HuR. It is also involved in the aberrant gene expression in different cancers. In this study, we have carried out a bioinformatics-based gene analysis and annotation of lincRNA-p21 to show that it is highly conserved in primates and identified two conserved domains in its sequence at the 5' and 3' terminal regions. hnRNPK has previously been shown to interact specifically with the 5' conserved region of lincRNA-p21. hnRNPK is known to bind preferentially to the pyrimidine-rich (poly C) nucleotide sequences in RNAs. Interestingly, we observed a single occurrence of a cytosine-rich patch (C-patch) consisting of a CUCCCGC sequence in the 5' conserved region of human lincRNA-p21, making it a putative hnRNPK binding motif. Using NMR and ITC experiments, we showed that the single-stranded C-patch containing RNA sequence motif interacts specifically with the KH3 domain of hnRNPK.

Structural and functional implications of leucine-rich repeats in toll-like receptor1 subfamily.

Leucine-rich repeats (LRRs) - the protein-protein and protein-ligand interaction motif of proteins participating in a plethora of functions in plants, vertebrates, invertebrates, and prokaryotes - are a fascinating piece of conserved yet versatile structural motif. In toll-like receptors (TLRs), this domain forms the extracellular part that is preceded by an intracellular toll/interleukin-1 receptor (TIR) domain. The extracellular part is crucial for recognizing a structurally diverse set of viral, bacterial, fungal, and parasite-derived components, while the TIR domain is recruited for activation of downstream signaling following recognition. The distinct ability of the paralogs TLR1 and TLR6 to dimerize with TLR2 and recognize different ligands intrigued and motivated us to exchange the dimerizing and ligand-binding residues between TLR1/6 and note the effect on dimer formation and ligand binding. The appreciable sequence modification brought about no significant alteration in the native scaffold of the motif, as revealed from the comparison of simulations with wild-type dimers. Moreover, docking of the exchanged ligands to the variant proteins supported favorable binding. Thus, the structural stability and the functional plasticity offered by the motif might be the reason for its extensive use across cellular functions and life forms, a feature crucial for coevolution and the knowledge essential for therapeutics.

Structure and DNA binding analysis of AT-rich interaction domain present in human BAF-B specific subunit BAF250b.

BAF250b and its paralog BAF250a are the DNA-binding central hub proteins present in BAF-B and BAF-A classes of SWI/SNF chromatin-remodeling complexes. BAF250b contains an AT-rich interaction domain (ARID) and C-terminal BAF250_C domain, and it is found mutated in several cancers. ARID is a conserved helix-turn-helix motif-containing DNA-binding domain present in several eukaryotic proteins. The ARID of BAF250b has been proposed to play roles in recruiting SWI/SNF to the target gene promoters for their activation. BAF250b ARID structures had been deposited in the protein data bank by a structural genomics consortium. However, it is not well-studied for its DNA-binding and solution dynamic properties. Here, we report complete backbone NMR resonance assignments of human BAF250b ARID. NMR chemical shifts and the backbone dynamics showed that the solution structure of the protein matched the reported crystal structures. The structure and chemical shift indexing revealed the presence of a short ß-sheet in the DNA-binding region of BAF250b ARID that was absent in the structure of its paralog BAF250a ARID. NMR chemical shift perturbations identified DNA-binding residues and revealed the DNA-binding interface on BAF250b ARID. NMR data-driven HADDOCK models of BAF250b ARID - DNA complexes revealed its plausible mode of DNA-binding. Isothermal titration calorimetry experiments showed that BAF250b ARID interacts with DNA sequences with moderate affinities like BAF250a ARID. However, distinct thermodynamic signatures were observed for binding of BAF250a ARID and BAF250b ARID to AT-rich DNA sequence, suggesting that subtle sequence and structural differences in these two proteins influence their DNA-binding.

Signatures of Specific DNA Binding by the AT-Rich Interaction Domain of BAF250a.

The AT-rich interaction domain (ARID) containing BAF250a is a subunit of the BAF-A class of SWI/SNF chromatin remodeling complexes. The ARID belongs to a family of conserved DNA binding domains found in several eukaryotic proteins; however, its exact contribution to BAF250a function and the mechanism of its DNA binding are not well understood. Here we have probed the interaction of the BAF250a ARID with three different double-stranded DNA (dsDNA) sequences to understand its DNA binding properties. A comprehensive biophysical and thermodynamic study using nuclear magnetic resonance (NMR) spectroscopy and isothermal titration calorimetry revealed the complex nature of BAF250a ARID-DNA interactions. The thermodynamic signatures of the BAF250a ARID with 12 A-T bp dsDNA (AT-12) are distinct from those of 12 G-C bp dsDNA (GC-12) or 12 bp Dickerson dodecamer DNA (DD-12) sequences. We observed that the binding of the BAF250a ARID with AT-12 DNA is enthalpically driven in a tested temperature range of 5-25 °C. BAF250a ARID/AT-12 DNA interaction exhibited a larger negative calorimetric specific heat change (ΔCp) compared to that of BAF250a ARID/GC-12 DNA or BAF250a ARID/DD-12 DNA interactions. In the presence of salt (NaCl), ARID/AT-12 DNA binding was less perturbed than ARID/GC-12 DNA or ARID/DD-12 DNA binding. Overall, these results show that BAF250a ARID/AT-12 DNA interaction has signatures of "specific" binding. Furthermore, using NMR chemical shift perturbation experiments, we have identified DNA binding residues on the BAF250a ARID and generated a data-driven HADDOCK model of the ARID/DNA complex that was further supported by mutating key lysine residues that were found to be important for DNA binding.

Molecular determinants of complex formation between DNA and the AT-rich interaction domain of BAF250a.

AT-rich interaction domain (ARID)-containing BAF250a protein is a central DNA-binding subunit of the SWI/SNF chromatin-remodeling complex. ARIDs are found in several eukaryotic proteins that play roles in different aspects of cellular physiology. However, despite their biological importance, ARIDs remain relatively uncharacterized for their dynamics and DNA binding. Here, we have probed the structure and DNA-binding properties of BAF250a ARID. We show that the core BAF250a ARID interacts with DNA sequences with low micromolar affinities. NMR chemical shift perturbation (CSP) results reveal a number of conserved residues in ARID that are involved in DNA binding. An NMR CSP-based docking model of ARID-DNA complexes reveals that BAF250a ARID possesses necessary determinants of specific DNA binding.

Domain architecture of BAF250a reveals the ARID and ARM-repeat domains with implication in function and assembly of the BAF remodeling complex.

BAF250a and BAF250b are subunits of the SWI/SNF chromatin-remodeling complex that recruit the complex to chromatin allowing transcriptional activation of several genes. Despite being the central subunits of the SWI/SNF complex, the structural and functional annotation of BAF250a/b remains poorly understood. BAF250a (nearly 2200 residues protein) harbors an N-terminal DNA binding ARID (~110 residues) and a C-terminal folded region (~250 residues) of unknown structure and function, recently annotated as BAF250_C. Using hydrophobic core analysis, fold prediction and comparative modeling, here we have defined a domain boundary and associate a ß-catenin like ARM-repeat fold to the C-terminus of BAF250a that encompass BAF250_C. The N-terminal DNA-binding ARID is found in diverse domain combinations in proteins imparting unique functions. We used a comparative sequence analysis based approach to study the ARIDs from diverse domain contexts and identified conserved residue positions that are important to preserve its core structure. Supporting this, mutation of one such conserved residue valine, at position 1067, to glycine, resulted in destabilization, loss of structural integrity and DNA binding affinity of ARID. Additionally, we identified a set of conserved and surface-exposed residues unique to the ARID when it co-occurs with the ARM repeat containing BAF250_C in BAF250a. Several of these residues are found mutated in somatic cancers. We predict that these residues in BAF250a may play important roles in mediating protein-DNA and protein-protein interactions in the BAF complex.

Mechanistic Insights into the Differential Catalysis by RheB and Its Mutants: Y35A and Y35A-D65A.

RheB GTPase is a Ras-related molecular switch, which regulates the mTOR signaling pathway by cycling between the active [guanosine triphosphate (GTP)] state and inactive [guanine diphosphate (GDP)] state. Impairment of GTPase activity because of mutations in several small GTPases is known to be associated with several cancers. The conventional GTPase mechanism such as in H-Ras requires a conserved glutamine (Q64) in the switch-II region of RheB to align the catalytic water molecule for efficient GTP hydrolysis. The conformation of this conserved glutamine is different in RheB, resulting in an altered conformation of the entire switch-II region. Studies on the atypical switch-II conformation in RheB revealed a distinct, noncanonical mode of GTP hydrolysis. An RheB mutant Y35A was previously shown to exclusively enhance the intrinsic GTPase activity of RheB, whereas the Y35A-D65A double mutant was shown to reduce the elevated GTPase activity. Here, we have used all-atom molecular dynamics (MD) simulations for comprehensive understanding of the conformational dynamics associated with the fast (Y35A) and slow (Y35A-D65A) hydrolyzing mutants of RheB. Using a combination of starting models from PDB structures and in-silico generated mutant structures, we discuss the observed conformational deviations in wild type (WT) versus mutants. Our results show that a number of interactions of RheB with phosphates of GTP as well as Mg2+ are destabilized in Y35A mutant in the switch-I region. We report distinct water dynamics at the active site of WT and mutants. Furthermore, principal component analysis showed significant differences in the conformational space sampled by the WT and mutants. Our observations provide improved understanding of the noncanonical GTP hydrolysis mechanism adopted by RheB and its modulation by Y35A and Y35A-D65A mutants.

Study of Q224K, V152G double mutation in bean PGIP2, an LRR protein for plant defense--an in silico approach.

Polygalacturonase inhibiting proteins (PGIPs) are leucine-rich repeat (LRR) proteins from plants that are organized into multigene families. They act as specific inhibitors against Polygalacturonases (PGs) from phytopathogens and share high sequence identity within species. We performed in silico mutation (Q224K and V152G) in PGIP2 from Phaseolus vulgaris to corresponding residues of another member, PGIP1. This mutation is known to cause 100% loss of inhibition against the PG of fungus Fusarium phyllophilum (Fp). A comparative analysis between PGIP2 and the double mutant, using 50 ns molecular dynamics simulations explored structural difference affecting PG binding properties. Simulations revealed that the mutation at 224, strains this residue which acts as a lock for the PGIP-PG complex through main chain H-bond. Changes in secondary structural elements and strain in the bend region along the convex face of the solenoidal protein affected the flexibility of the mutant protein. At the concave interacting face of the mutant, subtle changes in the sidechain behavior of the PG-binding residues occurred in a concerted manner revealing flipping of aromatic rings to be crucial to avoid steric clash with FpPG in PGIP2. Docking PGIP2 and the mutant protein individually to FpPG illustrated the inability of the latter to inhibit FpPG leaving its active site free. Our study demonstrates that the effect of mutation affects the flexibility of the protein along the convex face, while binding specificity is altered through the concave face imparting minimal change in the typical structure supported by the LRRs.

Comparative study of protein-protein interaction observed in PolyGalacturonase-Inhibiting Proteins from Phaseolus vulgaris and Glycine max and PolyGalacturonase from Fusarium moniliforme.

BACKGROUND: The PolyGalacturonase-Inhibiting Proteins (PGIP) of plant cell wall limit the invasion of phytopathogenic organisms by interacting with the enzyme PolyGalacturonase (PG) they secrete to degrade pectin present in the cell walls. PGIPs from different or same plant differ in their inhibitory activity towards the same PG. PGIP2 from Phaseolus vulgaris (Pv) inhibits the PG from Fusarium moniliforme (Fm) although PGIP1, another member of the multigene family from the same plant sharing 99% sequence similarity, cannot. Interestingly, PGIP3 from Glycine max (Gm) which is a homologue of PGIP2 is capable of inhibiting the same PG although the extent of similarity is lower and is 88%. It therefore appears that subtle changes in the sequence of plant PGIPs give rise to different specificity for inhibiting pathogenic PGs and there exists no direct dependence of function on the extent of sequence similarity. RESULTS: Structural information for any PGIP-PG complex being absent, we resorted to molecular modelling to gain insight into the mechanism of recognition and discrimination of PGs by PGIPs. We have built homology models of PvPGIP1 and GmPGIP3 using the crystal structure of PvPGIP2 (1OGQ) as template. These PGIPs were then docked individually to FmPG to elucidate the characteristics of their interactions. The mode of binding for PvPGIP1 to FmPG considerably differs from the mode observed for PvPGIP2-FmPG complex, regardless of the high sequence similarity the two PGIPs share. Both PvPGIP2 and GmPGIP3 despite being relatively less similar, interact with residues of FmPG that are known from mutational studies to constitute the active site of the enzyme. PvPGIP1 tends to interact with residues not located at the active site of FmPG. Looking into the electrostatic potential surface for individual PGIPs, it was evident that a portion of the interacting surface for PvPGIP1 differs from the corresponding region of PvPGIP2 or GmPGIP3. CONCLUSION: van der Waals and electrostatic interactions play an active role in PGIPs for proper recognition and discrimination of PGs. Docking studies reveal that PvPGIP2 and GmPGIP3 interact with the residues constituting the active site of FmPG with implications that the proteins bind/block FmPG at its active site and thereby inhibit the enzyme.