Biology subjects

Tama, F.

Publications and source records attributed to Tama, F..

4 recordsLinked to original sources

bioRxiv2025

Flexible fitting to infer atomistic-precision models of large-amplitude conformational dynamics in biomolecules from high-speed atomic force microscopy imaging

High-speed atomic force microscopy (HS-AFM) experiments allow direct observation of biomolecular dynamics at the single-molecule level, acquiring a large amount of topographic imaging data that visualizes changes of the molecular surface during functional activity over an extended period of time. Since images have no atomistic resolution, a major challenge has been to develop post-experimental computational methods to infer atomistic information from measurements. The recently developed NMFF-AFM flexible fitting method provides a computationally efficient approach promising to infer atomistic-precision models of conformational dynamics from resolution-limited AFM imaging data. We report the software integration of this method into the well-established BioAFMviewer platform and demonstrate first applications to experimental HS-AFM imaging data. To facilitate applications, we developed a direct workflow from raw experimental AFM data to visualization and analysis of fitting results. The presented applications to experimental data of a single protein domain, a protein complex, and a megadalton size protein filament demonstrate versatility of NMFF-AFM modelling to reproduce large-amplitude conformational motions of biomolecular dynamics from HS-AFM imaging. As a first step towards automated large-scale analysis of AFM imaging data, we furthermore demonstrate reconstruction of an atomistic molecular movie of protein dynamics, involving large-amplitude conformational transitions, from a measured HS-AFM movie sequence. Implementation of flexible fitting within the standalone user-friendly interactive BioAFMviewer software opens the opportunity for broad range applications to facilitate the understanding of resolution-limited HS-AFM measurements.

biophysics↗

bioRxiv2022

SWEET13 transport of sucrose, but not gibberellin, restores male fertility in Arabidopsis sweet13;14

SWEET sucrose transporters play important roles in the allocation of sucrose in plants. Some SWEETs were shown to also mediate transport of the plant growth regulator gibberellin (GA). The close physiological relationship between sucrose and GA raised the questions of if there is a functional connection, and whether one or both of the substrates are physiologically relevant. To dissect these two activities, molecular dynamics were used to map the binding sites of sucrose and GA in the pore of SWEET13 and predicted binding interactions that might be selective for sucrose or GA. Transport assays confirmed these predictions. In transport assays, the N76Q mutant had 7x higher relative GA3 activity, and the S142N mutant only transported sucrose. The impaired pollen viability and germination in sweet13;14 double mutants were complemented by the sucrose-selective SWEET13S142N but not by the SWEET13N76Q mutant, indicating that sucrose is the physiologically relevant substrate and that GA transport capacity is dispensable in the context of male fertility. Therefore, GA supplementation to counter male sterility may act indirectly via stimulating sucrose supply in male sterile mutants. These findings are also relevant in the context of the role of SWEETs in pathogen susceptibility.

plant biology↗

bioRxiv2019

Protein dynamics regulate distinct biochemical properties of cryptochromes in mammalian circadian rhythms

Circadian rhythms are generated by a transcription-based feedback loop where CLOCK:BMAL1 drive transcription of their repressors (PER1/2, CRY1/2), which bind to CLOCK:BMAL1 to close the feedback loop with ~24-hour periodicity. Here we identify a key biochemical and structural difference between CRY1 and CRY2 that underlies their differential strengths as transcriptional repressors. While both cryptochromes bind the BMAL1 transactivation domain with similar affinity to sequester it from coactivators, CRY1 is recruited with much higher affinity to the PAS domain core of CLOCK:BMAL1, allowing it to serve as a stronger repressor that lengthens circadian period. We identify a dynamic loop in the secondary pocket that regulates differential binding of cryptochromes to the PAS domain core. Notably, PER2 binding remodels this loop in CRY2 to enhance its affinity for CLOCK:BMAL1, explaining why CRY2 forms an obligate heterodimer with PER2, while CRY1 is capable of repressing CLOCK:BMAL1 both with and without PER2.

biochemistry↗

bioRxiv2019

Bipartite anchoring of SCREAM enforces stomatal initiation by coupling MAP Kinases to SPEECHLESS

Cell-fate in eukaryotes is regulated by MAP Kinases (MAPKs) that translate external cues to cellular responses. In plants, two MAPKs, MPK3/6, regulate diverse processes of development, environmental response, and immunity. Yet, the mechanism bridging these shared signaling components with a specific target remains unresolved. Focusing on the development of stomata, epidermal valves for gas exchange and transpiration, we report here that the bHLH protein SCREAM functions as a scaffold by recruiting MPK3/6 to downregulate SPEECHLESS, a transcription factor initiating stomatal cell lineages. SCREAM directly binds with MPK3/6 through an evolutionarily-conserved yet unconventional bipartite motif. Mutations in this motif abrogate association, phosphorylation and degradation of SCREAM, unmask hidden non-redundancies between MPK3 and MPK6, and result in uncontrolled stomatal differentiation. Structural analyses of MPK6 at the 2.75[A] resolution unraveled bipartite binding of SCREAM with MPK6, that is distinct from an upstream MAPKK. Our findings elucidate, at the atomic resolution, the mechanism directly linking extrinsic signals to transcriptional reprogramming during the establishment of stomatal cell-fate, and highlight a unique substrate-binding mode adopted by plant MAPKs.

plant biology↗