Biology subjects

Martin-Garcia, J. M.

Publications and source records attributed to Martin-Garcia, J. M..

5 recordsLinked to original sources

bioRxiv2025

Ambient temperature structural studies of Penicillin-binding Protein 2a of Methicillin-Resistant Staphylococcus aureus with XFELs and synchrotrons

Penicillin-binding protein 2a (PBP2a) is a transpeptidase responsible for the {beta}-lactam resistance in methicillin-resistant Staphylococcus aureus (MRSA), posing significant challenges to antibiotic therapy. PBP2as unique structural features, including its highly flexible active site and allosteric regulation, enable it to maintain catalytic activity even in the presence of {beta}-lactam antibiotics. Despite extensive characterization using cryogenic crystallography, key questions remain about its dynamic properties and conformational changes under near-physiological conditions. Room-temperature crystallography methods, particularly serial femtosecond X-ray crystallography (SFX) at XFELs, provide a powerful approach to capture these dynamics. Unlike cryogenic conditions that can constrain protein flexibility, SFX enables the study of conformational variability and interaction networks closer to physiological states. Here, we present the first room-temperature structures of PBP2a obtained using SFX to uncover insights into the enzymes flexibility, allosteric communication, and catalytic mechanisms. These findings are built upon optimized large-scale production and crystallization protocols for PBP2a, ensuring high-quality microcrystals suitable for SFX data collection. The room-temperature structures reveal novel interaction patterns, including unique salt bridge networks and dynamic structural elements absent in cryogenic studies. Furthermore, comparative analyses highlight how environmental conditions influence the conformational states of PBP2a, providing new perspectives on its resistance mechanisms. By integrating structural data from EuXFEL and LCLS, this study not only enhances our understanding of PBP2as functional dynamics but also underscores the value of room-temperature crystallography in studying antibiotic resistance. These insights could guide the design of next-generation {beta}-lactam antibiotics capable of overcoming PBP2a-mediated resistance.

biochemistry↗

bioRxiv2025

Active site tyrosine residues in human NQO1 homodimer are critical for non-synchronous enzyme catalysis at the two active sites.

AbstractHuman NQO1 is a flavoenzyme essential for the redox metabolization of many substances and associated with wide-impacting diseases such as cancer and Alzheime[r]s. Recent X-ray crystallographic studies have proposed that a few residues at the active site of NQO1 (including Tyr126 and Tyr128) may control enzyme catalysis and functional negative cooperativity. In this work, we use rapid mixing pre-steady state kinetics and hydrogen-deuterium exchange followed by mass spectrometry (HDX-MS) to evaluate experimentally the role of Tyr126 and Tyr128 in NQO1 functionality by generating mutants to Phe, Ala and Glu. Mutations to Phe caused mild effects, whereas those to Ala significantly decreased hydride transfer efficiency and those to Glu virtually abolished NQO1 activity. Interestingly, structural stability studies by HDX-MS showed significant perturbations particularly affecting the binding site of NADH/NAD+ in the less conservative mutations (particularly to Glu). Mutations of Tyr126 and Tyr128 seem to also modulate the non-synchronous catalysis in the two active sites (negative cooperativity) as well as the selectivity for NADH/NADPH as coenzymes. Our work experimentally demonstrates the critical role of Tyr126 and Tyr128 in the flavin reductive half-reaction of the catalytic cycle of NQO1 in the negative cooperativity, and also suggests that phosphorylation of these two Tyr residues might shut down NQO1 activity reversibly.

biophysics↗

bioRxiv2022

Structural and biophysical properties of FopA, a major outer membrane protein of Francisella tularensis

Francisella tularensis is an extremely infectious pathogen and a category A bioterrorism agent. It causes the highly contagious zoonosis, Tularemia. Currently, FDA approved vaccines against tularemia are unavailable. F. tularensis outer membrane protein A (FopA) is a well-studied virulence determinant and protective antigen against tularemia. It is a major outer membrane protein (Omp) of F. tularensis. However, FopA-based therapeutic intervention is hindered due to lack of complete structural information for membrane localized mature FopA. In our study, we established recombinant expression, monodisperse purification, crystallization and X-ray diffraction ([~]6.5 [A]) of membrane localized mature FopA. Further, we performed bioinformatics and biophysical experiments to unveil its structural organization in the outer membrane. FopA consists of 393 amino acids and has less than 40% sequence identity to known bacterial Omps. Using comprehensive sequence alignments and structure predictions together with existing partial structural information, we propose a two-domain organization for FopA. Circular dichroism spectroscopy and heat modifiability assay confirmed FopA has a {beta}-barrel domain consistent with alphafold2s prediction of an eight stranded {beta}-barrel at the N-terminus. Small angle X-ray scattering (SAXS) and native-polyacrylamide gel electrophoresis revealed FopA purified in detergent micelles is predominantly dimeric. Molecular density derived from SAXS at 31 [A] shows putative dimeric N-terminal {beta}-barrels surrounded by detergent corona and connected to C-terminal domains via flexible linker. Disorder analysis predicts N- and C-terminal domains are interspersed by a long intrinsically disordered region and alphafold2 predicts this region to be largely unstructured. Taken together, we propose a dimeric, two-domain organization of FopA in the outer membrane: the N-terminal {beta}-barrel is membrane embedded, provides dimerization interface and tethers to membrane extrinsic C-terminal domain via long flexible linker. Structure determination of membrane localized mature FopA is essential to understand its role in pathogenesis and develop anti-tularemia therapeutics. Our results pave the way towards it.

biochemistry↗

bioRxiv2020

New Structural Insights into the Function of the Catalytically Active Human Taspase1

Proteases can play essential roles in severe human pathology, ranging from degenerative and inflammatory illnesses to infectious diseases, with some, such as Taspase1, involved in growth and progression of tumors at primary and metastatic sites. Taspase1 is a N-terminal nucleophile (Ntn)-hydrolase overexpressed in primary human cancers, coordinating cancer cell proliferation, invasion, and metastasis. Loss of Taspase1 activity disrupts proliferation of human cancer cells in vitro and in mouse xenograft models of glioblastoma, thus this protein has the potential to become a novel anticancer drug target. It belongs to the family of Ntn-hydrolases, a unique family of proteins synthesized as enzymatically inactive proenzymes that become activated upon cleavage of the peptide bond on the N-terminal side of a threonine residue, which then becomes the catalytic site nucleophile. The activation process simultaneously changes the conformation of a long domain at the C-terminus of the alpha-subunit for which no full-length structural information exists and its function is poorly understood. Here we present a novel cloning strategy to generate a fully active, circularly permuted form of Taspase1 to determine the crystallographic structure of catalytically active human Taspase1 to 3.04[A]. We discovered that this region forms a long helical domain and is indispensable for the catalytic activity of Taspase1. Together, our study highlights the importance of this element for the enzymatic activity of Ntn-hydrolases and suggests that this long domain could be a novel target for the design of inhibitors with the potential to be developed into anticancer therapeutics.

biochemistry↗

bioRxiv2017

Authentic Enzyme Intermediates Captured "on-the-fly" by Mix-and-Inject Serial Crystallography

Ever since the first atomic structure of an enzyme was solved, the discovery of the mechanism and dynamics of reactions catalyzed by biomolecules has been the key goal for the understanding of the molecular processes that drive life on earth. Despite a large number of successful methods for trapping reaction intermediates, the direct observation of an ongoing reaction has been possible only in rare and exceptional cases. Here, we demonstrate a general method for capturing enzyme catalysis in-action by mix-and-inject serial crystallography. Specifically, we follow the catalytic reaction of the Mycobacterium tuberculosis -lactamase with the 3rd generation antibiotic ceftriaxone by time-resolved serial femtosecond crystallography. The results reveal, in near atomic detail, antibiotic cleavage and inactivation on the millisecond to second time scales including the crossover from transition state kinetics to steady-state kinetics.\n\nSynopsisAn enzymatically catalyzed reaction is initiated by diffusion based mixing of substrate and followed at runtime by time-resolved serial crystallography using a free electron laser.

biophysics↗