Dhamotharan, K. | bioRxiv Science

bioRxiv2024

EF-P and its paralog EfpL (YeiP) differentially control translation of proline containing sequences

Polyproline sequences (XPPX) stall ribosomes, thus being deleterious for all living organisms. In bacteria, translation elongation factor P (EF-P) plays a crucial role in overcoming such arrests. 12% of eubacteria possess an EF-P paralog - YeiP (EfpL) of unknown function. Here, we functionally and structurally characterize EfpL from Escherichia coli and demonstrate its yet unrecognized role in the translational stress response. Through ribosome profiling, we analyzed the EfpL arrest motif spectrum and discovered additional stalls beyond the canonical XPPX motifs at single-proline sequences (XPX), that both EF-P and EfpL can resolve. Notably, the two factors can also induce pauses. We further report that, contrary to the housekeeping EF-P, EfpL can sense the metabolic state of the cell, via lysine acylation. Together, our work uncovers a new player in ribosome rescue at proline-containing sequences, and provides evidence that co-occurrence of EF-P and EfpL is an evolutionary driver for higher bacterial growth rates.

microbiology↗

bioRxiv2022

Insight into the structural basis for dual nucleic acid - recognition by the Scaffold Attachment Factor B2 protein

The family of scaffold attachment factor B (SAFB) proteins comprises three members and was first identified as binders of the nuclear matrix/scaffold. Over the past two decades, SAFBs were shown to act in DNA repair, mRNA/(l)ncRNA processing, and as part of protein complexes with chromatin-modifying enzymes. SAFB proteins are approximately-100-kDa-sized dual nucleic acid-binding proteins with dedicated domains in an otherwise largely unstructured context, but whether and how they discriminate DNA- and RNA-binding has remained enigmatic. We here provide the SAFB2 DNA- and RNA-binding SAP and RRM domains in their functional boundaries and use solution NMR spectroscopy to ascribe DNA- and RNA-binding functions. We give insight into their target nucleic acid preferences and map the interfaces with respective nucleic acids on sparse data-derived SAP and RRM domain structures. Further, we provide evidence that the SAP domain exhibits intra-domain dynamics and a potential tendency to dimerise, which may expand its specifically targeted DNA sequence range. Our data provide a first molecular basis of and a starting point towards deciphering DNA- and RNA-binding functions of SAFB2 on the molecular level and serve a basis for understanding its localization to specific regions of chromatin and its involvement in the processing of specific RNA species.

biochemistry↗

bioRxiv2019

Structural role of essential light chains in the apicomplexan glideosome

AbstractApicomplexan parasites, such as Plasmodium falciparum and Toxoplasma gondii, traverse the host tissues and invade the host cells exhibiting a specific type of motility called gliding. The molecular mechanism of gliding lies in the actin-myosin motor localized to the intermembrane space between the plasma membrane and inner membrane complex (IMC) of the parasites. Myosin A (MyoA) is a part of the glideosome, a large multi-protein complex, which is anchored in the outer membrane of the IMC. MyoA is bound to the proximal essential light chain (ELC) and distal myosin light chain (MLC1), which further interact with the glideosome associated proteins GAP40, GAP45 and GAP50. Whereas structures of several individual glideosome components and small dimeric complexes have been solved, structural information concerning the interaction of larger glideosome subunits and their role in glideosome function still remains to be elucidated. Here, we present structures of a T. gondii trimeric glideosome sub complex composed of a myosin A light chain domain with bound MLC1 and TgELC1 or TgELC2. Regardless of the differences between the secondary structure content observed for free P. falciparum PfELC and T. gondii TgELC1 or TgELC2, the proteins interact with a conserved region of TgMyoA to form structurally conserved complexes. Upon interaction, the essential light chains undergo contraction and induce -helical structure in the myosin A C-terminus, stiffening the myosin lever arm. The complex formation is further stabilized through binding of a single calcium ion to T. gondii ELCs. Our work provides an important step towards the structural understanding of the entire glideosome and uncovering the role of its members in parasite motility and invasion. Author summaryApicomplexans, such as Toxoplasma gondii or the malaria agent Plasmodium falciparum, are small unicellular parasites that cause serious diseases in humans and other animals. These parasites move and infect the host cells by a unique type of motility called gliding. Gliding is empowered by an actin-myosin molecular motor located at the periphery of the parasites. Myosin interacts with additional proteins such as essential light chains to form the glideosome, a large protein assembly that anchors myosin in the inner membrane complex. Unfortunately, our understanding of the glideosome is insufficient because we lack the necessary structural information. Here we describe the first structures of trimeric glideosome sub complexes of T. gondii myosin A bound to two different light chain combinations, which show that T. gondii and P. falciparum form structurally conserved complexes. With an additional calcium-free complex structure, we demonstrate that calcium binding does not change the formation of the complexes, although it provides them with substantial stability. With additional data, we propose that the role of the essential light chains is to enhance myosin performance by inducing secondary structure in the C-terminus of myosin A. Our work represents an important step in unveiling the gliding mechanism of apicomplexan parasites.

biophysics↗