RNA plays a central role in all organisms and can fold into complex structures to orchestrate function. Visualization of such structures often requires crystallization, which can be a bottleneck in the structure-determination process. To promote crystallization, an RNA-recognition motif (RRM) of the U1A spliceosomal protein has been co-opted as a crystallization module. Specifically, the U1-snRNA hairpin II (hpII) single-stranded loop recognized by U1A can be transplanted into an RNA target to promote crystal contacts and to attain phase information via molecular replacement or anomalous diffraction methods using selenomethionine. Herein, we produced the F37M/F77M mutant of U1A to augment the phasing capability of this powerful crystallization module. Selenomethionine-substituted U1A(F37M/F77M) retains high affinity for hpII (KD of 59.7 ± 11.4 nM). The 2.20 Å resolution crystal structure reveals that the mutated sidechains make new S-π interactions in the hydrophobic core and are useful for single-wavelength anomalous diffraction. Crystals were also attained of U1A(F37M/F77M) in complex with a bacterial preQ1-II riboswitch. The F34M/F37M/F77M mutant was introduced similarly into a lab-evolved U1A variant (TBP6.9) that recognizes the internal bulged loop of HIV-1 TAR RNA. We envision that this short RNA sequence can be placed into non-essential duplex regions to promote crystallization and phasing of target RNAs. We show that selenomethionine-substituted TBP6.9(F34M/F37M/F77M) binds a TAR variant wherein the apical loop was replaced with a GNRA tetraloop (KD of 69.8 ± 2.9 nM), laying the groundwork for use of TBP6.9(F34M/F37M/F77M) as a crystallization module. These new tools are available to the research community.
Trichoderma spp. are considered as efficient biocontrol agent that can significantly reduces the growth of several soil borne and plant pathogens such as Rhizoctonia solani, Sclerotium rolfsii, Phythium aphanidermatium, Fusarium oxysporum in several mode of action. Trichoderma harzianum is one the important species in genus Trichoderma, which is capable of producing several effective lytic enzymes and antifungal antibiotics that compete to other fungal pathogens and promotes plant growth. The aim of present study is to predict and analyze the tertiary structure and their potential binding sites through bioinformatic tools and techniques. The protein sequences of enzymes were retrieved from UniProt database, followed by modelling of tertiary structure by Swiss-model Workspace and further validated using PROCHECK server which showed that from 75% to 90% residues are in favored region of Ramchandran Plot. These different validation steps proved that the predicted models are stable and it will provide an insight to its functional aspect which is based on tertiary structures. Furthermore, functional and conserved motifs were predicted through PROSITE database. These findings allow us to determine the protein families and domain which remain conserved throughout the evolution which may act as inducing or suppressing the biological activity of protein. Ligand binding site of enzymes has been predicted using SiteHound server by using four different chemical probes which allow us studying different ligand binding site. Thus, this study supported a scientific base for 3D structure modelling of lytic and defence enzymes and opens the new opportunities for further investigations in biological control of phytopathogens.
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