Biology subjects

Bagde, S. R.

Publications and source records attributed to Bagde, S. R..

3 recordsLinked to original sources

bioRxiv2021

Structure of a TRAPPII-Rab11 activation intermediate reveals GTPase substrate selection mechanisms

Rab1 and Rab11 are essential regulators of the eukaryotic secretory and endocytic recycling pathways. The TRAPP complexes activate these GTPases via nucleotide exchange using a shared set of core subunits. The basal specificity of the TRAPP core is towards Rab1, yet the TRAPPII complex is specific for Rab11. A steric gating mechanism has been proposed to explain TRAPPII counterselection against Rab1. Here we present cryoEM structures of the 22-subunit TRAPPII complex from budding yeast, including a TRAPPII-Rab11 nucleotide exchange intermediate. The Trs130 subunit provides a "leg" that positions the active site distal to the membrane surface, and this leg is required for steric gating. The related TRAPPIII complex is unable to activate Rab11 due to a repulsive interaction, which TRAPPII surmounts using the Trs120 subunit as a "lid" to enclose the active site. TRAPPII also adopts an open conformation enabling Rab11 to access and exit from the active site chamber.

cell biology↗

bioRxiv2021

Structures of core eukaryotic protein complexes

Protein-protein interactions play critical roles in biology, but despite decades of effort, the structures of many eukaryotic protein complexes are unknown, and there are likely many interactions that have not yet been identified. Here, we take advantage of recent advances in proteome-wide amino acid coevolution analysis and deep-learning-based structure modeling to systematically identify and build accurate models of core eukaryotic protein complexes, as represented within the Saccharomyces cerevisiae proteome. We use a combination of RoseTTAFold and AlphaFold to screen through paired multiple sequence alignments for 8.3 million pairs of S. cerevisiae proteins and build models for strongly predicted protein assemblies with two to five components. Comparison to existing interaction and structural data suggests that these predictions are likely to be quite accurate. We provide structure models spanning almost all key processes in Eukaryotic cells for 104 protein assemblies which have not been previously identified, and 608 which have not been structurally characterized. One-sentence summaryWe take advantage of recent advances in proteome-wide amino acid coevolution analysis and deep-learning-based structure modeling to systematically identify and build accurate models of core eukaryotic protein complexes.

biochemistry↗

bioRxiv2021

Filament dynamics driven by ATP hydrolysis modulates membrane binding of the bacterial actin MreB

MreB, the bacterial ancestor of eukaryotic actin, is responsible for shape in most rod-shaped bacteria. While the eukaryotic actin utilizes ATP hydrolysis to drive filament treadmilling, the relevance of nucleotide-driven polymerization dynamics for MreB function is unclear. Here, we report mechanistic insights into the interplay between nucleotide-binding, ATP hydrolysis and membrane-binding of Spiroplasma citri MreB5 (ScMreB5). Antiparallel double protofilament assembly of ScMreB5WT with ATP, ADP or AMPPNP and an ATPase deficient mutant ScMreB5E134A demonstrate that the filaments assemble independent of ATP hydrolysis. However, capture of the filament dynamics revealed that efficient filament formation, bundling through lateral interactions and filament disassembly are affected in ScMreB5E134A. Hence, the catalytic glutamate (Glu134 in ScMreB5) plays a dual role - it functions as a switch by sensing the ATP-bound state for filament assembly, and by assisting hydrolysis for triggering disassembly. Glu134 mutation also exhibits an allosteric effect on membrane binding, as observed from the reduced liposome binding compared to that of the wild type. Thus, ATP hydrolysis can modulate filament length and bundling, and consequently the orientation of MreB filaments on the cell membrane depending on the curvature. Binding of ScMreB5 with liposomes is mediated by surface charge-based interactions, demonstrating paralog and organism specific features for MreB function. We conclude that the conserved ATP-dependent polymerization and disassembly upon ATP hydrolysis has been repurposed for modulating curvature-dependent organization of filaments on the membrane.

biochemistry↗