Crystallography and Structural Biology. Cristalografia y Biologia Estructural (CBE)

Some most recent departmental results
(See also our remaining publications & projects)

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)

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)

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)

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)

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 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)



Do you want to see how protein molecules are? Join us!	The very first Acta Cryst. article was written by our predecessors... Acta Cryst. (1948) 1, 3-4 About our origins...

Some old departmental videos	CBE = Cristalografía y Biología Estructural Logotypes for: CBE Dept. + Institute + CSIC

We are pleased to announce that our colleague Prof Dr Santiago Garcia-Granda has been elected President of the International Union of Crystallography (IUCr) during its Twenty-Sixth General Assembly, 2023, held in Melbourne (Australia). Santiago García-Granda is full professor at the University of Oviedo (Spain), past President of the European Crystallographic Association (ECA), past President of the Spanish Crystallography and Crystal Growth Association (GE3C) and member of the Spanish National Committee for Crystallography.