Biology subjects

Malinauskaite, L.

Publications and source records attributed to Malinauskaite, L..

3 recordsLinked to original sources

bioRxiv2026

Dual recognition of phage SSB triggers DRT4 for antiphage defense

Defense-associated reverse transcriptase 4 (DRT4) protects bacteria against phages through single-stranded DNA (ssDNA) synthesis and activation of an effector RNase. How precisely phage recognition is coupled to RNase activation remains unclear. Here, we combine structural, biochemical, and genetic analyses to define the requirements for DRT4 activation. Cryo-electron microscopy structures capture DNA synthesis and provide a structural basis for preferential purine incorporation. Screening against the BASEL phage collection reveals protection against diverse phages and identifies the Bas17-encoded ssDNA-binding (SSB) protein gp44 as a DRT4 trigger. We show that DRT4 requires both recognition of its ssDNA product and direct protein-protein interactions of gp44 for RNase activation. Together, our findings support a previously unrecognized "two-factor authentication" model in which ssDNA-mediated recruitment of a phage SSB cooperates with specific protein interactions to license abortive infection for antiphage defense.

biochemistry↗

bioRxiv2020

Single-particle cryo-EM at atomic resolution

The three-dimensional positions of atoms in protein molecules define their structure and provide mechanistic insights into the roles they perform in complex biological processes. The more precisely atomic coordinates are determined, the more chemical information can be derived and the more knowledge about protein function may be inferred. With breakthroughs in electron detection and image processing technology, electron cryo-microscopy (cryo-EM) single-particle analysis has yielded protein structures with increasing levels of detail in recent years1,2. However, obtaining cryo-EM reconstructions with sufficient resolution to visualise individual atoms in proteins has thus far been elusive. Here, we show that using a new electron source, energy filter and camera, we obtained a 1.7 [A] resolution cryo-EM reconstruction for a prototypical human membrane protein, the {beta}3 GABAA receptor homopentamer3. Such maps allow a detailed understanding of small molecule coordination, visualisation of solvent molecules and alternative conformations for multiple amino acids, as well as unambiguous building of ordered acidic side chains and glycans. Applied to mouse apo-ferritin, our strategy led to a 1.2 [A] resolution reconstruction that, for the first time, offers a genuine atomic resolution view of a protein molecule using single particle cryo-EM. Moreover, the scattering potential from many hydrogen atoms can be visualised in difference maps, allowing a direct analysis of hydrogen bonding networks. Combination of the technological advances described here with further approaches to accelerate data acquisition and improve sample quality provide a route towards routine application of cryo-EM in high-throughput screening of small molecule modulators and structure-based drug discovery.

biophysics↗

bioRxiv2020

Role of transmembrane helix 6 in substrate recognition of the amino acid transporter MhsT

MhsT of Bacillus halodurans is a transporter of hydrophobic amino acids and a homologue of the eukaryotic SLC6 family of Na+-dependent symporters for amino acids, neurotransmitters, osmolytes, or creatine. The broad range of transported amino acids by MhsT prompted the investigation of the substrate recognition mechanism. Here, we report six new substrate-bound structures of MhsT, which, in conjunction with functional studies, reveal how the flexibility of a Gly-Met-Gly (GMG) motif in the unwound region of transmembrane segment 6 (TM6) is central for the recognition of substrates of different size by tailoring the binding site shape and volume. MhsT mutants, harboring substitutions within the unwound GMG loop and substrate binding pocket that mimick the binding sites of eukaryotic SLC6A18/B0AT3 and SLC6A19/B0AT1 transporters of neutral amino acids, exhibited impaired transport of aromatic amino acids that require a large binding site volume. Conservation of a general (G/A/C){Phi}G motif among eukaryotic members of SLC6 family suggests a role for this loop in a common mechanism for substrate recognition and translocation by SLC6 transporters of broad substrate specificity.

biochemistry↗