| Cristalografía
y Biología
Estructural
= CBE
|
Some
most recent results (See
also our remaining publications
& projects)
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|
 Successful participation of our
Department at the 44th SEBBM
Congress celebrated in Málaga:
Our colleague and "Ramón y Cajal" Fellow, Dr. Rafael Molina (left), presented his work developed in Denmark (NNF-CPR-University of Copenhagen) and received the 2nd MDPI-SEBBM Best poster Prize. Our PhD student Daniel Muñoz-Reyes (right), working under the supervision of Dr. María José Sánchez-Barrena was awarded with the IUBMB Best Short Talk Presentation Prize at the Protein Structure & Function Meeting celebrated in the Congress. Well done guys!  Lena Caruso joined us for a three months stay. She will be working on multicomponent crystallization on the supervision of Dr. Lourdes Infantes San Mateo
CBE news...  Join us!
 Logotypes: CBE Dept., CSIC & Institute CBE = Cristalografía y Biología Estructural  The very first Acta Cryst. article by our predecessors... Acta Cryst. (1948) 1, 3-4 About our origins...  |
 Dissecting
the role of PBP1 from S.
aureus, a Key Component of the Divisome Machinery.
The
Penicillin-Binding Proteins (PBPs) are transpeptidases that catalyze
crosslinking in the bacterial cell wall and the molecular targets of
the penicillin antibiotics. Here, we report an integrative structural
biology study of PBP1 from Staphylococcus
aureus
(saPBP1), providing mechanistic clues about its function and regulation
during cell division. A significant result provided in this work is the
unexpected arrangement of the PASTA domains respect to the rest PBP1
domains, pointing to profound functional implications. Thus, although
PBP1-PASTA domains preserve their compact fold, SAXS and
molecular-dynamics simulations revealed that they are conformationally
mobile and separated of transpeptidase domain in contrast with their
homologues in PBP2x from Streptococcus
pneumoniae and Streptococcus
thermophilus.
A series of crystallographic complexes with β-lactam
antibiotics
(as inhibitors) and penta-Glycine (as a substrate mimetic) allowed the
molecular characterization of both inhibition by antibiotics and
binding for the donor and acceptor peptidoglycan strands.
Mass-spectrometry experiments with synthetic peptidoglycan fragments
revealed binding by PASTA domains in coordination with the remaining
domains. Collectively, our structural and modelling studies allowed us,
for the first time, to decipher key structural features and differences
between saPBP1 and other PBPs. The observed mobility of the PASTA
domains in saPBP1 could play a crucial role for in vivo interaction
with its glycosyltransferase partner in the membrane or with other
components of the divisome machinery, as well as for coordination of
transpeptidation and polymerization processes in the bacterial divisome.
Computational and Structural Biotechnology Journal (2021) 19, 5392-5405 (doi: 10.1016/j.csbj.2021.09.018) 
The molecular players maintaining
plant cell membrane to face abiotic stress.
Cold, Salinity and other abiotic stresses cause crop productivity loss
at worldwide scale. Many of the physiological changes motivated by the
plant adaptation to these stresses unbalance lipid homeostasis at the
plasma membrane (PM) and induce membrane instability. To counteract
this situation, a highly controlled system drives lipid transfer from
the endoplasmic reticulum (ER) to PM through the so-called contact
sites. There, the Synaptotagmin protein family (SYTs) plays a central
role displaying a dual function to tether ER and PM and to transport
lipids from one membrane to the other. Our analysis explains how SYTs
function is activated by changes in the lipid composition at PM and
Ca2+ concentration at cytosol.
Life Science Alliance (2021) published on line 18 August (doi: 10.26508/lsa.202101152) Plant Cell (2021) 33, 2431-2453 (doi: 10.1093/plcell/koab122)  Molecular
mechanism behind
extremophilicity.
Endoxylanases active under extreme
conditions of temperature and alkalinity can replace the use of highly
pollutant chemicals in the pulp and paper industry. A comprehensive
bioinformatics study of the GH10 family identified Xyn11, with
extraordinary xylanolytic activity under extreme conditions. Addition
of a carbohydrate binding domain at the C-terminus of the protein
sequence further improved the activity of the enzyme at high pH. The
crystallographic analysis of Xyn11 structure provides an explanation of
its function at extreme conditions. Not surprisingly, the atomic
interactions responsible for Xyn11 resistance are the same known to
sustains protein stability, namely hydrogen bonds, ion pairs,
hydrophobic and aromatic interactions. However, the number and
distribution in which these interactions appear and its extremophilic
enzyme properties makes Xyn11 an outstanding case for study.
Computational and Structural Biotechnology Journal (2021) (doi: 10.1016/j.csbj.2021.05.004)  A
collaborative effort between scientist from the IQFR (Dept. of
Cristalografía y Biología Estructural and Dept.
of
Sistemas de Baja Dimensionalidad, Superficies y Materia Condensada) and
Dept. of Química Orgánica, Facultad de Ciencias
Químicas, Universidad Complutense of Madrid, allowed design,
synthesis and structural characterization of the first phasing agent
designed for solving native protein structures by X-ray crystallography
at the wavelength corresponding to the maximum intensity of the
synchrotron facilities applied in biomolecular crystallography.
The agent consists of a neutral ytterbium (III)-caged complex that
exhibits higher anomalous signals at shorter wavelengths when compared
to the best, currently applied lanthanide-based phasing agents. As a
proof of principle, the complex allows determining the 3D structure of
a 36 kDa protein without setting the incident beam wavelength at the
metal absorption edge, the strategy followed to date to gain the
strongest anomalous signal even at the expense of crystallographic
resolution. The agent becomes non-disruptive to the diffraction quality
of the marked crystals and allows determining accurate phases, both
leading to high-quality electron density maps that enable the full
tracing of the protein structure only with one agent unit bound to the
protein. The high-phasing power, efficient binding to the protein, low
metal−macromolecule ratio, and easy handling support the
developed Yb (III) complex as the best phasing agent for X-ray
crystallography of a complex biomacromolecule without using modified
analogues.
ACS Applied Bio Materials (2021) in press (doi: 10.1021/acsabm.1c00305)  Structural
cues for
understanding eEF1A2 moonlighting. Spontaneous mutations in
the EEF1A2 gene are increasingly being
recognized as a source of epilepsy and severe neurological disabilities
in children. The crystal structure of eEF1A2 protein purified from
rabbit skeletal muscle reveals the spatial locations of the side‐chain
carboxylates of Glu301 and Glu374, to which phosphatidylethanolamine is
uniquely attached via an amide bond, thereby defining the anchoring
points of an eEF1A2 dimer to cellular membranes. In this new light, the
role of eEF1A2 as an ancient, multifaceted, and articulated G protein
at the crossroads of autophagy, oncogenesis, and viral replication
appears very distant from the “canonical” one of
delivering aminoacyl‐tRNAs to the ribosome that has dominated the scene
and much of the thinking for many decades.
Chembiochem (2021) 22, 374 –391 (doi: 10.1002/cbic.202000516) |
The
molecular machinery behind processive fungal chitinases.
Chitinases degrade chitin, one of the most widespread polysaccharides
in nature, into low molecular weight chitooligomers (COS), which have a
broad range of pharmaceutical and medicinal applications. We report a
detailed picture of the molecular events behind substrate/products
binding in a fungal chitinase, giving full insight into its donor and
acceptor subsites. Our crystallographic analysis revealed a previously
unobserved dynamic on-off ligand binding process associated with motion
of its insertion CID domain. This might represent a molecular mechanism
complementary to the crucial role ascribed to aromatics at the
catalytic site in processivity, which is an essential property
modulating the bioconversion of chitin. Furthermore, our
analysis elucidates the implication of some highly flexible residues in
activity and suggested new targets to address engineering of these
biotechnologically important enzymes.
Computational and Structural Biotechnology Journal (2021) 19, 5466-5478 (doi: 10.1016/j.csbj.2021.09.027)  IUCrJ (2021) 8(6), published on line (doi: 10.1107/S2052252521008125) 
DDP1,
a
bridge between inositide and polyphosphate metabolisms. DDP1 is a yeast inositol
pyrophosphatase able to connect the inositide (InsP) and polyphosphate
(polyPs) metabolisms. In this work, recently published in Science
Advances and in collaboration with the University of Oxford, the
structure of DDP1 is analysed in complex with its multiple substrates
or analogues which belongs to three different type of compounds.
Particularly, this work uncovers the structural basis for inositide
pyrophosphates (PP-InsPs), dicucleotide phosphates (ApnAs) and
inorganic polyphosphates (polyPs) recognition. This protein is able to
clamp the phosphate groups of these substrates whereas the
pyrophosphate moiety to be cleaved remains near to the “Nudix
motif” responsible of the hydrolysis. This research revealed
a full view of DDP1 ligand binding and stability through a complete
analysis of DDP1 crystallographic structures, mutagenesis, thermal
shift and activity assays. The results of this work go beyond the
inositide signalling and reveal the molecular basis to design
inhibitors against a pathway strictly regulated in fungi, with putative
application in fungal infections treatment.
 A single residue swapping the
substrate ambiguity and chiral specificity in an esterase.
Carboxylic ester hydrolases are among the most important biocatalysts
in the field of biotechnology because of their capacity to catalyze
hydrolysis with exquisite enantio-, regio-, and stereospecificity. We
previously identified EH3, which has remarkable multi-specificity, with
sequence positions that modulate both substrate ambiguity and chiral
specificity. Here, combined analyses of specificity through
evolutionary trace, structure determination and mutagenesis reveal that
substrate ambiguity and chiral specificity in a single hydrolase can be
modulated by a single residue. In this way, it is feasible to engineer
prominent substrate-promiscuous yet stereospecific hydrolases that are
relevant to the field of organic synthesis. The work is a collaborative
study with the ICP (CSIC), with the participation of
scientist from USA and UK.
Computational and Structural Biotechnology Journal (2021) (doi: 10.1016/j.csbj.2021.04.041)  Collaboration
between IQFR scientists, CBM Severo Ochoa center and the Vigo and A
Coruña Universities, together with the synthetic chemical
company Galchimia, reveals unprecedent characteristics in a thermophile
esterase, obtained from a hot spring metagenomic library.
Esterases and lipases count
with keen
industrial interest due to its
capacity to accept a wide range of non-natural substrates and stability
in organic solvents, and, in the case of extremophile organisms, its
ability to tolerate extreme temperatures and pH. In this work,
published in the Computational and Structural Biotechnology Journal, a
novel esterase, EstD11, has been biochemically and structurally
characterized. The tridimensional structure of EstD11 and the inactive
mutant has been determined together with 8 crystallographic complexes
with different substrates and products of the esterase reaction,
revealing the molecular basis underlying enzyme reactivity and
specificity. Interestingly, a unique methionine zipper has been
identified lining the active site and cap domains that may be
responsible for the thermostability and catalytic promiscuity of the
protein. Furthermore, the success of high-temperature crystallization
experiments has been key in obtaining singular information on cap
dynamics.
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