Biology subjects

Mechulam, Y.

Publications and source records attributed to Mechulam, Y..

7 recordsLinked to original sources

bioRxiv2026

Coordinated Assembly of WAVE and WASH complexes

WAVE and WASH polymerize Arp2/3-mediated branched actin at the leading edge of migrating cells and on the surface of endosomes, respectively. These two proteins are regulated within similar multiprotein complexes, the assembly mechanism of which remains poorly understood. Here we found using mass spectrometry that the two smallest subunits, BRK1 of the WAVE complex and CCDC53 of the WASH complex, interact with each other and with the assembly factor HSBP1. HSBP1 promotes WAVE and WASH assemblies and their respective activities, migration persistence and endosomal branched actin. Reduced levels of WAVE and WASH complexes caused by HSBP1 depletion can be corrected by providing cells with an excess of BRK1 or CCDC53 to assemble their specific complex. Unexpectedly, endogenous levels of BRK1 cross-regulate WASH assembly, whereas those of CCDC53 cross-regulate WAVE assembly. We found that various oligomers comprising BRK1, CCDC53 and HSBP1, including a heterotrimer containing one molecule of each, are formed through their promiscuous coiled coils. These oligomers play a coordinating role in the assembly of WAVE and WASH complexes.

cell biology↗

bioRxiv2025

A new family of ribosome hibernation factors in Archaea

Ribosome hibernation preserves translation machinery during stress, yet its mechanisms in Archaea remain poorly defined. Here we identify Hib, a previously unrecognized family of hibernation factors widespread in Archaea. Deletion of hib in Thermococcus barophilus delays recovery from stationary phase and reduces 70S ribosome pools, establishing its role in ribosome preservation. Hib displays a unique modular architecture, combining a bacterial-like HPF/RaiA domain with a Cystathionine Beta Synthase module. High-resolution cryo-EM structures of reconstituted and in cell-extracted Hib:ribosome complexes from Pyrococcus abyssi identify three conformations encompassing the positions of tRNAs at A, P and E sites during translation. Thus, Hib acts as a hibernation factor blocking all states of the dynamic translation process, preserving the ribosome from dissociation and degradation. Our findings define Hib as a key hibernation factor in Archaea and provide a framework for understanding ribosome dormancy and adaptation across all domains of life. Beyond the discovery of Hib, a comprehensive phylogenetic analysis highlights the evolutionary relationships between prevalent ribosome hibernation factors across Bacteria and Archaea.

microbiology↗

bioRxiv2024

Cryo-EM Structures of Saccharolobus solfataricus Initiation Complexes with Leaderless mRNAs Highlight Archaeal Features and Eukaryotic Proximity

The archaeal ribosome is of the eukaryotic type. Genomic and phylogenetic studies have indicated that TACK and Asgard, the closest relatives of eukaryotes, have ribosomes containing eukaryotic ribosomal proteins not found in other archaeal branches, eS25, eS26 and eS30. In our study, we investigated the case of Saccharolobus solfataricus, a crenarchaeon belonging to the TACK branch, which mainly uses leaderless mRNAs. We characterized the small ribosomal subunit of S. solfataricus bound to SD-leadered or leaderless mRNAs (lmRNAs). Cryo-EM structures show for the first time archaeal versions of eS25, eS26 and eS30 proteins bound to the small subunit. In addition, we identify two novel ribosomal proteins named aS33 and aS34 as well as a domain of eS6, that highlight the diversity of archaeal ribosomes. Leaderless mRNAs are bound to the small ribosomal subunit, and the 5-triphosphate group contributes to their binding. Archaeal eS26 is in the mRNA exit channel wrapped around the 3 end of ribosomal RNA, as it is in eukaryotes. Its position is not compatible with an SD:antiSD duplex in the mRNA exit channel. Overall, our results suggest a role of eS26 in translation regulation and possible evolutionary routes from archaeal to eukaryotic translation.

molecular biology↗

bioRxiv2023

Visualization of translation reorganization upon persistent collision stress in mammalian cells

Aberrantly slow mRNA translation leads to ribosome stalling and subsequent collision with the trailing neighbor. Ribosome collisions have recently been shown to act as stress sensors in the cell, with the ability to trigger stress responses balancing survival and apoptotic cell-fate decisions depending on the stress level. However, we lack a molecular understanding of the reorganization of translation processes over time in mammalian cells exposed to an unresolved collision stress. Here we visualize the effect of a persistent collision stress on translation using in situ cryo electron tomography. We observe that low dose anisomycin collision stress leads to the stabilization of Z-site bound tRNA on elongating 80S ribosomes, as well as to the accumulation of an off-pathway 80S complex possibly resulting from collision splitting events. We visualize collided disomes in situ, occurring on compressed polysomes and revealing a stabilized geometry involving the Z-tRNA and L1 stalk on the stalled ribosome, and eEF2 bound to its collided rotated-2 neighbor. In addition, non-functional post-splitting 60S complexes accumulate in the stressed cells, indicating a limiting Ribosome associated Quality Control clearing rate. Finally, we observe the apparition of tRNA-bound aberrant 40S complexes shifting with the stress timepoint, suggesting a succession of different initiation inhibition mechanisms over time. Altogether, our work visualizes the changes of translation complexes under persistent collision stress in mammalian cells, indicating how perturbations in initiation, elongation and quality control processes contribute to an overall reduced protein synthesis. SummaryUsing in situ cryo electron tomography we visualized the reorganization of mammalian translation processes during a persistent collision stress.

cell biology↗

bioRxiv2022

Redesigning methionyl-tRNA synthetase for β-methionine activity with adaptive landscape flattening and experiments

Amino acids (AAs) with a noncanonical backbone would be a valuable tool for protein engineering, enabling new structural motifs and building blocks. To incorporate them into an expanded genetic code, the first, key step is to obtain an appropriate aminoacyl-tRNA synthetase (aaRS). Currently, directed evolution is not available to optimize such AAs, since an appropriate selective pressure is not available. Computational protein design (CPD) is an alternative. We used a new CPD method to redesign MetRS and increase its activity towards {beta}-Met, which has an extra backbone methylene. The new method considered a few active site positions for design and used a Monte Carlo exploration of the corresponding sequence space. During the exploration, a bias energy was adaptively learned, such that the free energy landscape of the apo enzyme was flattened. Enzyme variants could then be sampled, in the presence of the ligand and the bias energy, according to their {beta}-Met binding affinities. Eleven predicted variants were chosen for experimental testing; all exhibited detectable activity for {beta}-Met adenylation. Top predicted hits were characterized experimentally in detail. Dissociation constants, catalytic rates, and Michaelis constants for both -Met and {beta}-Met were measured. The best mutant retained a preference for -Met over {beta}-Met; however, the preference was reduced, compared to the wildtype, by a factor of 29. For this mutant, high resolution crystal structures were obtained in complex with both -Met and {beta}-Met, indicating that the predicted, active conformation of {beta}-Met in the active site was retained. Author summaryAmino acids (AAs) with a noncanonical backbone would be valuable for protein engineering, enabling new structural motifs. To incorporate them into an expanded genetic code, the key step is to obtain an appropriate aminoacyl-tRNA synthetase (aaRS). Currently, directed evolution is not available to optimize such AAs. Computational protein design is an alternative. We used a new method to redesign MetRS and increase its activity towards {beta}-Met, which has an extra backbone methylene. The method considered a few active site positions for design and used a Monte Carlo exploration of sequence space, during which a bias energy was adaptively learned, such that the free energy landscape of the apo enzyme was flattened. Enzyme variants could then be sampled, in the presence of the ligand and the bias energy, according to their {beta}-Met binding affinities. Eleven predicted variants were chosen for experimental testing; all exhibited detectable {beta}-Met adenylation activity. Top hits were characterized experimentally in detail. The best mutant had its preference for -Met over {beta}-Met reduced by a factor of 29. Crystal structures indicated that the predicted, active conformation of {beta}-Met in the active site was retained.

biochemistry↗

bioRxiv2022

Role of aIF5B in archaeal translation initiation

In eukaryotes and in archaea late steps of translation initiation involve the two initiation factors e/aIF5B and e/aIF1A. In eukaryotes, the role of eIF5B in ribosomal subunit joining is established and structural data showing eIF5B bound to the full ribosome were obtained. To achieve its function, eIF5B collaborates with eIF1A. However, structural data illustrating how these two factors interact on the small ribosomal subunit have long been awaited. The role of the archaeal counterparts, aIF5B and aIF1A, remains to be extensively addressed. Here, we study the late steps of Pyrococcus abyssi translation initiation. Using in vitro reconstituted initiation complexes and light scattering, we show that aIF5B bound to GTP accelerates subunit joining without the need for GTP hydrolysis. We report the crystallographic structures of aIF5B bound to GDP and GTP and analyze domain movements associated to these two nucleotide states. Finally, we present the cryo-EM structure of an initiation complex containing 30S bound to mRNA, Met-tRNAiMet, aIF5B and aIF1A at 2.7 [A] resolution. Structural data shows how archaeal 5B and 1A factors cooperate to induce a conformation of the initiator tRNA favorable to subunit joining. Archaeal and eukaryotic features of late steps of translation initiation are discussed.

biochemistry↗

bioRxiv2019

Adaptive landscape flattening allows the design of both enzyme:substrate binding and catalytic power

Designed enzymes are of fundamental and technological interest. Experimental directed evolution still has significant limitations, and computational approaches are complementary. A designed enzyme should satisfy multiple criteria: stability, substrate binding, transition state binding. Such multi-objective design is computationally challenging. Two recent studies used adaptive importance sampling Monte Carlo to redesign proteins for ligand binding. By first flattening the energy landscape of the apo protein, they obtained positive design for the bound state and negative design for the unbound. We extend the method to the design of an enzyme for specific transition state binding, i.e., for catalytic power. We consider methionyl-tRNA synthetase (MetRS), which attaches methionine (Met) to its cognate tRNA, establishing codon identity. MetRS and other synthetases have been extensively redesigned by experimental directed evolution to accept noncanonical amino acids as substrates, leading to genetic code expansion. We redesigned MetRS computationally to bind several ligands: the Met analog azidonorleucine, methionyl-adenylate (MetAMP), and the activated ligands that form the transition state for MetAMP production. Enzyme mutants known to have azidonorleucine activity were recovered, and mutants predicted to bind MetAMP were characterized experimentally and found to be active. Mutants predicted to have low activation free energies for MetAMP production were found to be active and the predicted reaction rates agreed well with the experimental values. We expect the present method will become the paradigm for computational enzyme design.

biochemistry↗