tRNA shape is an identity element for an archaeal pyrrolysyl-tRNA synthetase from the human gut.

Protein translation is orchestrated through tRNA aminoacylation and ribosomal elongation. Among the highly conserved structure of tRNAs, they have distinguishing features which promote interaction with their cognate aminoacyl tRNA synthetase (aaRS). These key features are referred to as identity elements. In our study, we investigated the tRNA:aaRS pair that installs the 22nd amino acid, pyrrolysine (tRNAPyl:PylRS). Pyrrolysyl-tRNA synthetases (PylRSs) are naturally encoded in some archaeal and bacterial genomes to acylate tRNAPyl with pyrrolysine. Their large amino acid binding pocket and poor recognition of the tRNA anticodon have been instrumental in incorporating >200 noncanonical amino acids. PylRS enzymes can be divided into three classes based on their genomic structure. Two classes contain both an N-terminal and C-terminal domain, however the third class (ΔpylSn) lacks the N-terminal domain. In this study we explored the tRNA identity elements for a ΔpylSn tRNAPyl from Candidatus Methanomethylophilus alvus which drives the orthogonality seen with its cognate PylRS (MaPylRS). From aminoacylation and translation assays we identified five key elements in ΔpylSn tRNAPyl necessary for MaPylRS activity. The absence of a base (position 8) and a G-U wobble pair (G28:U42) were found to affect the high-resolution structure of the tRNA, while molecular dynamic simulations led us to acknowledge the rigidity imparted from the G-C base pairs (G3:C70 and G5:C68).

Enzymes known as PylRS offer the remarkable ability to expand the natural genetic code of a living cell with unnatural amino acids. Currently, over 200 unnatural amino acids can be genetically encoded with the help of PylRS and its partner tRNAPyl, enabling us to endow proteins with novel properties, or regulate protein activity using light or inducible cross-linking. One intriguing feature of PylRS enzymes is their ability to avoid cross-reactivity when two PylRS homologs from different organisms-such as those from the archaea Methanosarcina mazei and Methanomethylophilus alvus-are co-expressed in a single cell. This makes it possible to simultaneously encode two unnatural amino acids in a single protein. This study illuminates the elusive mechanism of PylRS specificity by using cryo-electron microscopy, biochemistry and molecular simulations. The interaction of PylRS from M. alvus with its tRNAPyl is best described as two pieces of a jigsaw puzzle; in which PylRS recognizes the unique shape of its cognate tRNA instead of specific nucleotides in the tRNA sequence like other tRNA-binding enzymes. This finding may streamline the rational design of tools for simultaneous genetic incorporation of multiple unnatural amino acids, thereby facilitating the development of valuable proteins for research, medicine, and biotechnology.

Identifying the molecular basis of Laminin N-terminal domain Ca2+ binding using a hybrid approach.

Ca2+ is a highly abundant ion involved in numerous biological processes, particularly in multicellular eukaryotic organisms where it exerts many of these functions through interactions with Ca2+ binding proteins. The laminin N-terminal (LN) domain is found in members of the laminin and netrin protein families where it plays a critical role in the function of these proteins. The LN domain of laminins and netrins is a Ca2+ binding domain and in many cases requires Ca2+ to perform its biological function. Here, we conduct a detailed examination of the molecular basis of the LN domain Ca2+ interaction combining structural, computational, bioinformatics, and biophysical techniques. By combining computational and bioinformatic techniques with x-ray crystallography we explore the molecular basis of the LN domain Ca2+ interaction and identify a conserved sequence present in Ca2+ binding LN domains. These findings enable a sequence-based prediction of LN domain Ca2+ binding ability. We use thermal shift assays and isothermal titration calorimetry to explore the biophysical properties of the LN domain Ca2+ interaction. We show that the netrin-1 LN domain exhibits a high affinity and specificity for Ca2+, which structurally stabilizes the LN domain. This study elucidates the molecular foundation of the LN domain Ca2+ binding interaction and provides a detailed functional characterization of this essential interaction, advancing our understanding of protein-Ca2+ dynamics within the context of the LN domain.

Investigation of dynamic solution interactions between NET-1 and UNC-5B by multi-wavelength analytical ultracentrifugation.

NET-1 is a key chemotropic ligand that signals commissural axon migration and change in direction. NET-1 and its receptor UNC-5B switch axon growth cones from attraction to repulsion. The biophysical properties of the NET-1 + UNC-5B complex have been poorly characterized. Using multi-wavelength-AUC by adding a fluorophore to UNC-5B, we were able to separate the UNC-5B sedimentation from NET-1. Using both multi-wavelength- and single-wavelength AUC, we investigated NET-1 and UNC-5B hydrodynamic parameters and complex formation. The sedimentation velocity experiments show that NET-1 exists in a monomer-dimer equilibrium. A close study of the association shows that NET-1 forms a pH-sensitive dimer that interacts in an anti-parallel orientation. UNC-5B can form equimolar NET-1 + UNC-5B heterocomplexes with both monomeric and dimeric NET-1.