Summary of some most recent departmental results
See also our remaining publications & projects)
Bulgecin. Click to get a larger image
Structural characterization of lytic transglycosylase MltD of Pseudomonas aeruginosa, a target for the natural product bulgecin A. The natural inhibitor bulgecin A potentiates the activity of β-lactam antibiotics through the inhibition of three lytic transglycosylases in Pseudomonas aeruginosa (MltD, Slt and MltG). A recent study, led by Juan A. Hermoso from the Institute of Physical Chemistry “Blas Cabrera” and published in the International Journal of Biological Macromolecules, reports three X-ray structures that shed light on the structure of the multimodular MltD enzyme and provides a detailed molecular recognition of bulgecin A. These structures reveal a unique combination of a catalytic module and four cell-wall binding LysM modules: one unpredicted LysM module tightly attached to the catalytic domain while others exhibit mobility. A ternary complex structure provides two independent structures. One delineates the expansive active-site cleft of MltD by the insertion of a helical region, a hallmark of family 1D of lytic transglycosylases, providing a handbook explanation of the endolytic reaction by MltD. The other elucidates the mechanism of the exolytic reaction, demonstrating how the substrate’s terminal anhydro-NAM moiety is sequestered at subsite +2. These findings offer a comprehensive understanding of MltD’s role in cell-wall turnover events, elucidating both endolytic and exolytic activities, and provide insight into the molecular recognition of bulgecin A, paving the way for the development of more effective inhibitors.
International Journal of Biological Macromolecules (2024)  (doi: 10.1016/j.ijbiomac.2024.131420)

Proteobacteria. Click to get a larger image
Unique cross-link types in Alpha and Betaproteobacteria: A distinctive family of L,D-transpeptidases catalyzing L-Ala-mDAP crosslinks in Alpha-and Betaproteobacteria. The bacterial cell-wall peptidoglycan is made of glycan strands cross-linked by short peptide stems. Two different cross-link types have been long known, 4,3 and 3,3 cross-links, catalyzed by PBP transpeptidases and LD transpeptidases respectively. A multidimensional work led by Felipe Cava from Umeå University (Sweden), in collaboration with Juan A. Hermoso from the Institute of Physical Chemistry “Blas Cabrera” has explored novel cross-link types found in Alpha- and Betaproteobacteria, adding a new dimension to our understanding of bacterial cell-wall architecture. The study, published in Nature Communications, identifies a LD-transpeptidase from Gluconobacter oxydans, LDTGo, capable of generating 1,3 cross-links. LDTGo-like proteins have also been found among Alpha- and Betaproteobacteria, that lacks LD 3,3 transpeptidases. A high-resolution structure of LDTGo has been determined, revealing distinctive features including a Proline-rich region that limits substrate access, and a cavity for accommodating both glycan and peptide stem and responsible for the substrate specificity. These unique properties highlight the diversity of LD transpeptidases. Furthermore, the study demonstrates the dependence of 1,3 cross-link formation on substrate availability, involving the function of a DD endopeptidase. This discovery opens new avenues for understanding cell-wall integrity and maintenance in bacteria, particularly among Alpha and Betaproteobacteria.
Nature Communications (2024) 15, 1343  (doi: 10.1038/s41467-024-45620-5)

Division-septum. Click to get a larger image
In a collaborative effort with the groups of Lok-To Sham and Luo Min (National Univ. of Singapore) and Juan A. Hermoso (IQF-CSIC) the machinery of cell division FtsEX:RipC has been elucidated. The FtsEX complex regulates, directly or via a protein mediator depending on bacterial genera, peptidoglycan degradation for cell division. Here we report our investigation of Mycobacterium tuberculosis FtsEX as a non-canonical regulator with high basal ATPase activity. The cryo-EM structures of the FtsEX system alone and in complex with RipC, as well as the ATP-activated state, unveil detailed information on the signal transduction mechanism, leading to the activation of RipC. Our ļ¬ndings indicate that RipC is recognized through a “Match and Fit” mechanism, resulting in an asymmetric rearrangement of the extracellular domains of FtsX and a unique inclined binding mode of RipC. This study provides insights into the molecular mechanisms of FtsEX and RipC regulation in the context of a critical human pathogen, guiding the design of drugs targeting peptidoglycan remodeling.
Nature Communications (2023) published online  (doi: 10.1038/s41467-023-43770-6)

BlaC. Click on it to get a larger image
XFELs to reveal the heterogeneity in M. tuberculosis β-lactamase inhibition by Sulbactam. This work builds on possibilities unleashed by mix-and-inject serial crystallography at XFELs. We have triggered an enzymatic reaction by mixing an inhibitor with enzyme microcrystals to report, in atomic detail and at room temperature, how the Mycobacterium tuberculosis enzyme BlaC is inhibited by sulbactam. Our results reveal ligand binding heterogeneity, ligand gating, cooperativity, induced fit, and conformational selection, detailing how the inhibitor approaches the catalytic clefts and binds to the enzyme noncovalently before reacting to a trans-enamine.
Nature Communications (2023) (doi: 10.1038/s41467-023-41246-1)


AnmK. Click on it to get a larger image
Catalytic process of anhydro-N-acetylmuramic acid kinase (AnmK) from Pseudomonas aeruginosa. Peptidoglycan is a rigid envelope surrounding the cytoplasmic membrane of most bacterial species. The enzymatic processes that build, remodel, and recycle the chemical components of this cross-linked polymer are preeminent targets of antibiotics. In a collaborative effort with the group of Shahriar Mobashery and Amr M. El-Araby (Univ. Notre Dame), and Juan A. Hermoso (IQF-CSIC) we report a comprehensive kinetic and structural analysis for one such enzyme, the Pseudomonas aeruginosa anhydro-N- acetylmuramic acid (anhNAM) kinase (AnmK). AnmK follows a random-sequential kinetic mechanism with respect to its anhNAM and ATP substrates. Crystallographic analyses of four distinct structures demonstrate that both substrates enter the active site independently in an ungated conformation of the substrate subsites, with protein loops acting as gates for anhNAM binding. A remarkable X-ray structure for dimeric AnmK sheds light on the pre-catalytic and post-catalytic ternary complexes, one in each subunit. Computational simulations in conjunction with the four high-resolution X-ray structures reveal the full catalytic cycle.
Journal of Biological Chemistry (2023)  (doi: 10.1016/j.jbc.2023.105198)

LytB. Clck on it to get a larger image
Decoding the Molecular Basis of the Cell Division Final Step in Streptococcus pneumoniae. Bacterial cell-wall hydrolases must be tightly regulated during bacterial cell division to prevent aberrant cell lysis and to allow final separation of viable daughter cells. In a multidisciplinary work, we disclose the molecular dialogue between the cell-wall hydrolase LytB, wall teichoic acids, and the eukaryotic-like protein kinase StkP in Streptococcus pneumoniae. After characterizing the peptidoglycan recognition mode by the catalytic domain of LytB, we further demonstrate that LytB possesses a modular organization allowing the specific binding to wall teichoic acids and to the protein kinase StkP. Structural and cellular studies notably reveal that the temporal and spatial localization of LytB is governed by the interaction between specific modules of LytB and the final PASTA domain of StkP. Our data collectively provide a comprehensive understanding of how LytB performs final separation of daughter cells and highlights the regulatory role of eukaryotic-like kinases on lytic machineries in the last step of cell division in streptococci. Considering that LytB is recognized as a virulence factor involved in different aspects of host infection and that the pneumococcus is on the WHO list of priority pathogens for research and development of new antibiotics, our work holds the promise of providing a structural basis for the rational design of new drugs to combat pneumococcal infections.
Cell Reports (2023)   (doi: 10.1016/j.celrep.2023.112756)

Click on the image to get a larger copy
Structure-guided engineering of a receptor-agonist pair for inducible activation of the ABA adaptive response to drought.
Abscisic acid (ABA) is a plant hormone that naturally controls the response of plants in drought situations. Based on the atomic structure of ABA receptor proteins, we have designed a synthetic ABA receptor and a small chemical compound that acting together in plants are capable of activating ABA signaling in plants and very efficiently improving their tolerance to drought.
Science Advances (2023) 9(10)   (doi: 10.1126/sciadv.ade9948)   (see video 1) (see video 2)

Cyclosporin. Click on it to get a larger image
Structural Basis for Cyclosporin Isoform-Specific Inhibition of Cyclophilins from Toxoplasma gondii.
Cyclosporin (CsA) has antiparasite activity against the human pathogen Toxoplasma gondii. In a collaborative effort between University of Verona and the IQFR we characterized the functional and structural properties of two cyclophilins from T. gondii, TgCyp23 and TgCyp18.4. While TgCyp23 is a highly active cis−trans-prolyl isomerase (PPIase) and binds CsA with nanomolar affinity, TgCyp18.4 shows low PPIase activity and is significantly less sensitive to CsA inhibition. The crystal structure of the TgCyp23:CsA complex was solved at 1.1 Å resolution showing the molecular details of CsA recognition by the protein, and revealing relevant differences at the CsA-binding site compared to TgCyp18.4. The biochemical and structural data presented herein represents a relevant step toward understanding the molecular mechanisms of the anti-Toxoplasma action of CsA and may be instrumental in the rational design of new therapeutic drugs modulating TgCyp activity

ACS Infectious Diseases (2023)   (doi: 10.1021/acsinfecdis.2c00566)

IP3K. Click on it to get a larger image
IP3K, the enzyme that metabolizes the second messenger IP3, exhibits unexpected activity on carbohydrate-based ligands and on those displaying primary hydroxyls in the reactive position. Inositol 1,4,5-trisphosphate (IP3) is a second messenger that triggers the release of intracellular Ca2+. The Ca2+ signals cease when IP3 is metabolized, primarily by the enzyme IP3 3-kinase (IP3K). This enzyme converts IP3 into Inositol 1,3,4,5-tetrakisphosphate (IP4) and is crucial for processes such as memory, the immune system, and tumor progression, making it an attractive target for cancer research.
We have led a study on IP3K in collaboration with Prof. Barry V.L. Potter from the University of Oxford and Dr. Charles A. Brearley from the University of East Anglia. This study broadens our understanding of the biosynthetic capabilities of IP3K beyond its natural substrate, IP3, despite its notable specificity. We have revealed that IP3K exhibits plasticity, conferring tolerance to IP3-derived ligands with modifications, mainly at positions 1 and 3 of the inositol ring. Moreover, the study characterizes the IP3K activity against unexpected ligands, particularly those based on carbohydrates, and those that modify the reactive 3-position from a secondary to primary hydroxyls. To achieve this, we have used X-ray crystallography in combination with multiple techniques including chemical synthesis, fluorescence anisotropy, HPLC or computational docking.

These discoveries enhance our understanding of the IP3K family and the inositol polyphosphate metabolism, which are crucial for multiple cell functions. Furthermore, they will aid in the design of selective ligands against different IP3 targets,  with potential applications in cancer research.
Nature Communications (2024) 15, 1502  (doi: 10.1038/s41467-024-45917-5)

p6 nucleocomplex, clik to get a larger image
The genome-organizing protein p6 of Bacillus subtilis bacteriophage φ29 plays an essential role in viral development by activating the initiation of DNA replication and participating in the early-to-late transcriptional switch. These activities require the formation of a nucleoprotein complex in which the DNA adopts a right-handed superhelix wrapping around a multimeric p6 scaffold, restraining positive supercoiling and compacting the viral genome. Due to the absence of homologous structures, prior attempts to unveil p6's structural architecture failed. Here, we employed AlphaFold2 to engineer rational p6 constructs yielding crystals for three-dimensional structure determination. Our findings reveal a novel fold adopted by p6 that sheds light on its self-association mechanism and its interaction with DNA. By means of protein–DNA docking and molecular dynamic simulations, we have generated a comprehensive structural model for the nucleoprotein complex that consistently aligns with its established biochemical and thermodynamic parameters. Besides, through analytical ultracentrifugation, we have confirmed the hydrodynamic properties of the nucleocomplex, further validating in solution our proposed model. Importantly, the disclosed structure not only provides a highly accurate explanation for previously experimental data accumulated over decades, but also enhances our holistic understanding of the structural and functional attributes of protein p6 during φ29 infection.
Nucleic Acids Research (2024)  (doi: 10.1093/nar/gkae041

NCS1-Ric8. Click to get a larger image
The neuronal calcium sensor NCS-1 regulates the phosphorylation state and activity of the Gα chaperone and GEF Ric-8A.The Neuronal Calcium Sensor 1, an EF-hand Ca2+ binding protein, and Ric-8A coregulate synapse number and probability of neurotransmitter release. The protein-protein interaction interface constitutes a pharmacological target under brain pathological conditions. Previous structural studies of Ric-8A bound to Gα have revealed how Ric-8A phosphorylation promotes Gα recognition and activity as a chaperone and guanine nucleotide exchange factor. However, the molecular mechanism by which NCS-1 regulates Ric-8A activity and its interaction with Gα subunits was not well understood. Here, we have conducted a multimodal approach to show that NCS-1 and Ric-8A constitute a hub that integrates Ca2+, phosphorylation and G-protein signaling. The emergent picture indicates that at Ca2+ resting state, Ric-8A activity is under NCS-1 control and the Ca2+ sensor traps Ric-8A in a conformational state that hinders phosphorylation and Gα recognition. However, a specific Ca2+ signal triggers the disassembly of the NCS-1/Ric-8A complex, which in turn allows phosphorylation of Ric-8A, formation of the Ric-8A/Gα complex and activation of Gα nucleotide exchange. Strikingly, we found that NCS-1 binds Na+ in its regulatory Ca2+ site, decreasing the affinity of NCS-1 for Ca2+. Furthermore, we show that different Ca2+ signals promote the recognition of Ric-8A and dopamine D2 receptor. Finally, the high-resolution crystallographic data reported here define the NCS-1/Ric-8A interface and will allow the development of therapeutic synapse function regulators with improved activity and selectivity.
eLife (2023) 12:e86151  (doi: 10.7554/eLife.86151)

NQO1. Click on it to get a larger image
New structural insight into the conformational heterogeneity of NQO1 enzyme with XFELs. NQO1 is a flavoenzyme essential for the antioxidant defense system, stabilization of tumor suppressors, and the NAD(P)H-dependent two-electron reduction of a wide variety of substrates, including the activation of quinone-based chemotherapeutics. In addition, alterations in NQO1 function are associated with cancer, Alzheimer's and Parkinson's disease, which makes this enzyme an attractive target for drug discovery. The results reported in this article demonstrate the power of the SFX technique with XFELs to describe the structure-function relationship in NQO1. We provide important insight into the conformational heterogeneity of the human NQO1, highlighting the high plasticity of this enzyme in the catalytic site and hence shed light on the molecular basis of NQO1 functional cooperativity.
Lab on a Chip (2023) (doi: 10.1039/D3LC00176H)

DipM. Click on it to get a larger image
DipM controls multiple autolysins and mediates a regulatory feedback loop promoting cell constriction in Caulobacter crescentus. A multidimensional investigation led by Martin Thanbichler from the University of Marburg and in collaboration with Juan A. Hermoso (Institute of Physical Chemistry “Blas Cabrera”),  has identified two critical regulatory hubs that control cell division in Caulobacter crescentus, a widely studied bacterium. Published in Nature Communications, the research reveals the pivotal role of DipM and LdpF in coordinating peptidoglycan remodeling pathways, ensuring proper cell constriction and daughter cell separation. The findings offer valuable insights into bacterial growth and potential targets for the development of effective antibacterial drugs.
Nature Communications (2023) 14, Article number: 4095  (doi: 10.1038/s41467-023-39783-w)

FtsEXEnvCAmiB. Click on the image to get a larger copy
Mechanistic insights into the regulation of cell wall hydrolysis. The bacterial division is an essential cellular process that involves the formation of a septum made of peptidoglycan. The septum is initially shared between daughters and must be processed to complete division. Septal splitting has long been known to be mediated by enzymes called amidases that are controlled by an activator protein and the ABC-transporter- like complex called FtsEX. However, the mechanism of amidase regulation by this system has remained unclear. In a collaborative effort with the groups of Luo Min and Chris Sam (Univ. Singapore), Thomas Bernhardt (Harvard Univ.) and Juan A. Hermoso (IQF-CSIC), we report the structure of FtsEX in complex with an amidase and amidase activator, revealing how ATP binding to the complex promotes amidase activation and providing structural information that may help target the activation mechanism for the development of cell lysis-inducing antibiotics.
Proceedings of the National Academy of Sciences (2023)
 (doi: 10.1073/pnas.2301897120)
See also two short movies: ATP binding leading to PLD restraining and EnvC activation caused by the restraining of PLD upon ATP binding

Sis0455 Click on the ikmage to get a larger copy
Deciphering the Second Messenger Processing Mechanism by Standalone CRISPR-Cas Ring Nucleases.
CRISPR-Cas systems comprise an adaptive immune system in bacteria and archaea against foreign mobile genetic elements, such as plasmids and phages, which has constituted a revolution in life sciences. Their discovery and straightforward development into versatile nucleases by guide RNA exchange paved the way for gene modifications à la carte that can be employed in biomedicine and biotechnology.

Type III CRISPR-Cas effector systems detect foreign RNA triggering DNA and RNA cleavage and synthesizing cyclic oligoadenylate molecules (cA) in their Cas10 subunit. cAs act as a second messenger activating auxiliary nucleases, leading to an indiscriminate RNA degradation that can end in cell dormancy or death. Standalone ring nucleases are CRISPR ancillary proteins which downregulate the strong immune response of Type III systems by degrading cA. Two genes with this function (Sis0811 and Sis0455) have been found within the Sulfolobus islandicus (Sis) genome. They code for a long polypeptide composed by a CARF domain fused to an HTH domain (Sis0811 described in Molina et al., Nucleic Acids Research, 2021) and a short polypeptide constituted by a CARF domain with a 40 residue C-terminal insertion (Sis0455). Here, we determine the structure of the apo and substrate bound states of the Sis0455 enzyme, revealing an insertion at the C-terminal region of the CARF domain, which plays a key role closing the catalytic site upon substrate binding. Our analysis reveals the key residues of Sis0455 during cleavage and the coupling of the active site closing with their positioning to proceed with cA4 phosphodiester hydrolysis. A time course comparison of cA4 cleavage between the short, Sis0455, and long ring nucleases, Sis0811, shows the slower cleavage kinetics of the former, suggesting that the combination of these two types of enzymes with the same function in a genome could be an evolutionary strategy to regulate the levels of the second messenger in different infection scenarios.
Nucleic Acids Research (2022)   (doi: 10.1093/nar/gkac923)

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