Biology subjects

Garcia-Alles, L. F.

Publications and source records attributed to Garcia-Alles, L. F..

4 recordsLinked to original sources

bioRxiv2025

Promiscuous structural cross-compatibilities between major shell components of Klebsiella pneumoniae bacterial microcompartments

Bacterial microcompartments (BMC) are submicrometric reactors that encapsulate dedicated metabolic activities. BMC-H hexamers, the most abundant components of BMC shells, play major roles for shell plasticity and permeability. In part, chemical exchanges between the BMC lumen and the cellular cytosol will be defined by the disposition of amino acids lining the central BMC-H pores. Current models attribute to BMC-H a homo-oligomeric nature. The hexagonal symmetry of corresponding pores, however, would break down if hetero-hexamers formed, a possibility suggested by the frequent presence of multiple paralogs within BMC operons. Here, we gauged the degree of structural promiscuity between the 11 BMC-H paralogs from Klebsiella pneumoniae, a potential human pathogen endowed with the capacity to express three different BMC types. Concomitant activation of transcription of several BMC operons was first shown to be possible. By leveraging an adapted tripartite GFP technology, all possible BMC-H pair combinations were screened in E. coli. Multiple structural cross-compatibilities were pinpointed between homologs arising not only from the same BMC operon, but also from different BMC types, results supported by Alphafold and ESMFold predictions. The structural stability and assembly propensity of selected hetero-associations was established by biochemical means. In light of these results, we reinterpreted published lysine cross-linking mass spectrometry data to demonstrate that one of these hetero-hexamers, involving PduA and PduJ, was already detected to form in the shell of a recombinantly-expressed 1,2-propanediol utilization compartment from Salmonella enterica. Altogether, this study points to the need to embrace an augmented structural complexity in BMC shells.

biochemistry↗

bioRxiv2024

Designing symmetrical multi-component proteins using a hybrid generative AI approach

Proteins, the fundamental building blocks of biological function, orchestrate complex cellular processes by assembling into intricate structures through meticulous interactions. The design of specific protein-protein interfaces to create customized protein assemblies holds immense potential for various biotechnological applications. To address the current limitations in designing heteromeric interactions for multi-component assemblies, we developed a hybrid generative AI design approach. This method combines deep learning and automated reasoning, explicitly considering both positive and negative interaction states to favor heteromeric desired over undesired interactions. The approach leverages Effie, a deep-learned pairwise decomposable scoring function, and an advanced reasoning tool extended for multicriteria optimization of this function. Here, we tested the ability of this hybrid AI method to redesign homomeric interfaces of bacterial microcompartment components (BMC-H) into heteromeric assemblies. We benchmarked its performance against ProteinMPNN, a sequence design autoregressive model. Our in silico assessment, complemented by experimental validation, highlights the outperformance of the hybrid AI generative design approach, and its potential to unlock the engineering of complex multi-component self-assembling protein entities.

bioengineering↗

bioRxiv2024

Combining systems and synthetic biology for in vivo enzymology

Enzymatic parameters are classically determined in vitro, under conditions that are far from those encountered in cells, casting doubt on their physiological relevance. We developed a generic approach combining tools from synthetic and systems biology to measure enzymatic parameters in vivo. In the context of a synthetic carotenoid pathway in Saccharomyces cerevisiae, we focused on a phytoene synthase and three phytoene desaturases, which are difficult to study in vitro. We designed, built, and analyzed a collection of yeast strains mimicking substantial variations in substrate concentration by strategically manipulating the expression of geranyl-geranyl pyrophosphate (GGPP) synthase. We successfully determined in vivo Michaelis-Menten parameters (KM, Vmax and kcat) for GGPP-converting phytoene synthase from absolute metabolomics, fluxomics and proteomics data, highlighting differences between in vivo and in vitro parameters. Leveraging the versatility of the same set of strains, we then extracted enzymatic parameters for two of the three phytoene desaturases. Our approach demonstrates the feasibility of assessing enzymatic parameters directly in vivo, providing a novel perspective on the kinetic characteristics of enzymes in real cellular conditions.

systems biology↗

bioRxiv2019

Occurrence and stability of hetero-hexamer associations formed by β-carboxysome CcmK shell components

The carboxysome is a bacterial micro-compartment (BMC) subtype that encapsulates enzymatic activities necessary for carbon fixation. Carboxysome shells are composed of a relatively complex cocktail of proteins, their precise number and identity being species dependent. Shell components can be classified in two structural families, the most abundant class associating as hexamers (BMC-H) that are supposed to be major players for regulating shell permeability. Up to recently, these proteins were proposed to associate as homo-oligomers. Genomic data, however, demonstrated the existence of paralogs coding for multiple shell subunits. Here, we studied cross-association compatibilities among BMC-H CcmK proteins of Synechocystis sp. PCC6803. Co-expression in Escherichia coli proved a consistent formation of hetero-hexamers combining CcmK1 and CcmK2 or, remarkably, CcmK3 and CcmK4 subunits. Unlike CcmK1/K2 hetero-hexamers, the stoichiometry of incorporation of CcmK3 in associations with CcmK4 was low. Cross-interactions implicating other combinations were weak, highlighting a structural segregation of the two groups that could relate to gene organization. Sequence analysis and structural models permitted to localize interactions that would favor formation of CcmK3/K4 hetero-hexamers. Attempts to crystallize these CcmK3/K4 associations conducted to the unambiguous elucidation of a CcmK4 homo-hexamer structure. Yet, subunit exchange could not be demonstrated in vitro. Biophysical measurements showed that hetero-hexamers are thermally less stable than homo-hexamers, and impeded in forming larger assemblies. These novel findings are discussed in frame with reported data to propose a functional scenario in which minor CcmK3/K4 incorporation in shells would introduce sufficient local disorder as to allow shell remodeling necessary to adapt rapidly to environmental changes.

biochemistry↗