Unit
STRUCTURE OF MACROMOLECULAR TARGETS
Welcome to the Structure of Macromolecular Targets Unit!
Our research focuses on understanding the three-dimensional structure of proteins, their assembly into cellular macromolecular complexes, and the mechanisms underlying their function and regulation. We are particularly interested in how protein malfunctions impact our health, identifying disease-causing protein variants, and developing new therapeutic strategies to correct or bypass defective protein activity.
We employ a multidisciplinary approach that combines structural, biochemical, biophysical, and cellular methods to define the shape, function, and evolution of these macromolecular targets and map their “social network”. We use all possible techniques to get answers and ask better questions.Joining our lab means being trained in this interdisciplinary mindset and acquiring diverse technical skills in an environment of hard work and collaboration.
Santiago Ramón-Maiques (Santi)
Principal Investigator
Santiago, born in Valencia in 1973, earned his PhD in Biology in 2001 from the University of Valencia, under the guidance of Prof. Vicente Rubio at the Instituto de Biomedicina de Valencia (IBV). Trained in Biochemistry, Enzymology and Structural Biology, he specialized in X-ray crystallography with Prof. Ignacio Fita (CBMB, Barcelona). In this period, he contributed to the discovery of a new structural group of enzymes: the amino acid kinase family. Driven by a passion for understanding molecular machines and large macromolecular complexes, Santiago joined Dr. Wei Yang‘s lab at the National Institute of Health (NIH, Bethesda, USA) from 2003 to 2008, studying challenging proteins involved in DNA replication, recombination and repair. During this time, he also collaborated part-time with Dr. Alasdair Steven’s group (NIAMS, NIH), applying single-particle electron microscopy to study DNA-protein complexes that were inaccessible through X-ray crystallography. In 2010, Santiago returned to Spain as a Junior Group Leader at the Spanish National Cancer Research Centre (CNIO, Madrid), where he was awarded a “Ramón y Cajal” research contract. After seven years at CNIO, he became “Científico Titular” at CSIC and moved his group to the Centro de Biología Molecular Severo Ochoa (CBMSO, Madrid). In 2020, his lab relocated to IBV, where he now serves as Deputy Director and collaborates with the CIBERER on rare disease research. Outside the lab, Santiago enjoys spending time with his family, the sea, the waves and the wind. He is also a terrible but enthusiastic guitar player.
Francisco del Caño (Paco)
Postdoctoral Fellow
Maria Luisa López (Marisa)
Postdoctoral Fellow
Marisa (Toledo, 1977) earned her degree in Biochemistry from the University of Córdoba in 2001. She then moved to the Insituto de Biomedicina de Valencia (IBV) to dor her PhD in the field of X-ray crystallography under the guidance of Prof. Alberto Marina Moreno. During her PhD, she focused mainly on Two-Component Systems (TCS), dedicating her work to both the biochemical characterization of new systems and the structural resolution of key TCS proteins. After graduating in 2011, Marisa continued her training with a postdoctoral period in the United States. She joined David L. Stokes’ group at the Skirball Institute of NYULMC in New York. During this time, she specialized in solving membrane protein structures using cryo-electron microscopy (cryoEM). Her primary focus was on studying membrane proteins involved in various diseases, such as a zinc transporter called YiiP. Throughout this period, she gained expertise in handling lipids, detergents, nanodiscs, reconstructing membrane proteins within these systems, and subsequently characterizing and solving their structures using cryoEM. Likewise, she became proficient in all cryoEM techniques from sample preparation to data processing using classic software such as RELION and CryoSPARC. After 10 years (January 2021), she had the opportunity to return to Spain to establish the first cryoEM service in Valencia from the ground up, within José Luis Llácer’s group at the IBV. During the first four years, she set up all the necessary computing infrastructure and software required for cryoEM data processing and structure determination. Simultaneously, she developed protocols and conducted training sessions for sample preparation, both for negative stain and cryoEM. Since January 2025, she joined Santiago Ramón-Maiques’ laboratory at the, assisting the group in launching their cryoEM studies. Her work involves setting up and managing computational programs as well as optimizing the conditions for grid preparation in cryoEM. Besides frying proteins with electrons , she likes partying and scuba diving.
Lluís Eixerés
PhD student
Lluís is a second-year PhD student in Santiago’s lab. He earned his Bachelor’s degree in Biochemistry and Biomedical Sciences from the University of Valencia in 2021, followed by a Master’s in Research and Development in Biotechnology and Biomedicine from the same institution in 2022. During his undergraduate years, Lluís gained hands-on experience as an intern in the Department of Biochemistry and Molecular Biology under the guidance of Dr. Patricia Casino. There, he mastered fundamental molecular biology and microbiology techniques and became proficient in protein expression and purification. Additionally, he completed a paid internships at the Chair of Science Communication at the University of Valencia, where he developed expertise in scientific outreach and journalism. Lluís’s master’s thesis focused on the structural and functional bases of phosphotransfer systems in the pathogenic fungus Candida albicans. His dedication to the project led to continued work post-graduation, culminating in a publication in Communications Biology. In 2023, Lluís embarked on his PhD journey, aiming to unravel the unknown function of uracil phosphoribosyltransferase (UPRT) in humans and other higher eukaryotes.His research employs a multidisciplinary approach, including X-ray crystallography and cryo-electron microscopy, biochemical assays, and cellular techniques to study subcellular localization and generate CRISPR-KO cell lines. Along the way, he has contributed to other lab projects and co-authored a recent publication in the Journal of Molecular Biology. Since joining the lab, Lluís has presented his work at different meetings, including the 1st annual joint scientific conference CIPF-IBV, where he gave the inaugural lecture, and the ICAP/ICAN 2024 international meeting in Kaiserslautern (Germany), where he presented an oral communication. He also attended the renowned Diamond-CCP4 Data Collection & Structure Solution Workshop 2023 in Oxford, a premier course in protein crystallography with hands-on practice at the synchrotron beamlines. Outside the lab, Lluís moonlights as a semi-pro tennis player and a TV show gladiator, though, for now, science remains the only job that reliably pays the rent.
Carolina Espinosa (Carol)
Technician
Macromolecular pyrimidine factories
Pyrimidine nucleotides are vital for all life forms, serving as key components of nucleic acids (DNA and RNA) and acting as activators in glycosylation and the synthesis of carbohydrates and phospholipids. Cells acquire pyrimidine nucleotides via two distinct metabolic pathways. Slowly dividing or differentiated cells rely on dietary intake or nucleic acid breakdown through salvage pathways, while proliferating cells, including cancerous ones, synthesize pyrimidines de novo (from scratch). Our research aims to unravel the complex protein machinery responsible for pyrimidine biosynthesis. Mutations in these proteins lead to severe diseases, while inhibitors could serve as potential anti-tumor agents. But which proteins are involved, and how do they function?
At the core of this process is CAD, a multifunctional protein that fuses four enzymatic domains: glutaminase (GLN), carbamoyl phosphate synthetase (CPS-2), aspartate transcarbamoylase (ATC), and dihydroorotase (DHO). CAD assembles into large hexameric particles that drive pyrimidine synthesis. Despite its critical role, CAD’s structure and function remain elusive. We employ protein engineering, X-ray crystallography, and electron microscopy, combined with biochemical and cellular assays, to elucidate CAD’s structure and its catalytic and regulatory mechanisms. Additionally, we investigate other proteins involved in both de novo and salvage pyrimidine biosynthesis pathways.

Identifying and understanding CAD pathogenic changes
Mutations in CAD lead to a rare epileptic encephalopathy that predominantly affects newborns and young children. This severe neurometabolic disorder can lethal, but patients respond remarkably well to a treatment with oral supplements of uridine, making early diagnosis crucial. Our lab has developed a rapid and reliable cellular assay to assess the disease-causing potential of CAD mutations. Through global collaborations with hospitals, we assist in accurately diagnosing affected children and identifying those who could benefit from uridine therapy.

Once we identified a pathogenic variant, we return to the lab bench to study the impact of these mutations. By analyzing the damaging changes, we gain insights into CAD’s functional mechanisms. This knowledge is vital for understanding the molecular basis of the disease and predicting the pathogenic potential of future mutations.
Disease-causing mutations on proteins and protein networks
A protein’s function relies heavily on its structure and interactions with other proteins or molecules. Some mutations have an obvious impact, such as altering an active site residue, while others affect distant regions with unclear consequences. When a protein’s structure or function is poorly understood, evaluating the effects of mutations becomes even more challenging. As a lab specializing in solving protein structures and understanding their function, we leverage our expertise to help assessing the damaging potential of protein variants of uncertain significance. We aim to identify pathogenic changes, elucidate their mechanisms and develop new therapeutic strategies to restore faulty protein function and combat disease.
High conformational flexibility of phosphomannomutase 2: Implications for functioning mechanisms, stability and pharmacological chaperone design. Francisco del Caño-Ochoa, Marçal Vilar, Alicia Vilas, Rebeca Company, Belén Pérez, Santiago Ramón-Maiques. bioRxiv 2025.01.27.635082; doi: https://doi.org/10.1101/2025.01.27.635082
Synthetic heparan sulfate mimics based on chitosan derivatives show broad-spectrum antiviral activity. Revuelta J, Rusu L, Frances-Gomez C, Trapero E, Iglesias S, Pinilla EC,Blázquez AB, Gutiérrez-Adán A, Konuparamban A, Moreno O, Gómez Martínez M, Forcada-Nadal A, López-Redondo ML, Avilés-Alía AI; IBV-Covid19-Pipeline; Llácer JL, Llop J, Martín Acebes MÁ, Geller R, Fernández-Mayoralas A. Commun Biol. 2025, 8(1):360.
Disruption of CAD Oligomerization by Pathogenic Variants. Del Caño-Ochoa F, Ramadane-Morchadi L, Eixerés L, Moreno-Morcillo M,Fernández-Leiro R, Ramón-Maiques S. J Mol Biol. 2024, 436(23):168832.
Significance of utilizing in silico structural analysis and phenotypic data to characterize phenylalanine hydroxylase variants: A PAH landscape. Himmelreich N, Ramón-Maiques S, Navarrete R, Castejon-Fernandez N, Garbade SF, Martinez A, Desviat LR, Pérez B, Blau N. Mol Genet Metab. 2024 Jul;142(3):108514.
PAH deficient pathology in humanized c.1066-11G>A phenylketonuria mice. Martínez-Pizarro A, Picó S, López-Márquez A, Rodriguez-López C, Montalvo E,Alvarez M, Castro M, Ramón-Maiques S, Pérez B, Lucas JJ, Richard E, Desviat LR. Hum Mol Genet. 2024, 33(12):1074-1089.
Biallelic hypomorphic variants in CAD cause uridine-responsive macrocytic anaemia with elevated haemoglobin-A2. Steinberg-Shemer O, Yacobovich J, Noy-Lotan S, Dgany O, Krasnov T, Barg A,Landau YE, Kneller K, Somech R, Gilad O, Brik Simon D, Orenstein N, Izraeli S,Del Caño-Ochoa F, Tamary H, Ramón-Maiques S. Br J Haematol. 2024, 204(3):1067-1071.
Beyond genetics: Deciphering the impact of missense variants in CAD deficiency. Del Caño-Ochoa F, Ng BG, Rubio-Del-Campo A, Mahajan S, Wilson MP, Vilar M,Rymen D, Sánchez-Pintos P, Kenny J, Ley Martos M, Campos T, Wortmann SB, Freeze HH, Ramón-Maiques S. J Inherit Metab Dis. 2023 Nov;46(6):1170-1185.
A tailored strategy to crosslink the aspartate transcarbamoylase domain of the multienzymatic protein CAD. Del Caño-Ochoa F, Rubio-Del-Campo A, Ramón-Maiques S. Molecules. 2023, 28(2):660.
Pathogenic variants of the coenzyme A biosynthesis-associated enzyme phosphopantothenoylcysteine decarboxylase cause autosomal-recessive dilated cardiomyopathy. Bravo-Alonso I, Morin M, Arribas-Carreira L, Álvarez M, Pedrón-Giner C,Soletto L, Santolaria C, Ramón-Maiques S, Ugarte M, Rodríguez-Pombo P, Ariño J,Moreno-Pelayo MÁ, Pérez B. J Inherit Metab Dis. 2023, 46(2):261-272.
Phosphorylation of T897 in the dimerization domain of Gemin5 modulates protein interactions and translation regulation. Francisco-Velilla R, Embarc-Buh A, Abellan S, Del Caño-Ochoa F, Ramón-Maiques S, Martinez-Salas E. Comput Struct Biotechnol J. 2022, 20:6182-6191.
A functional platform for the selection of pathogenic variants of PMM2 amenable to rescue via the use of pharmacological chaperones. Segovia-Falquina C, Vilas A, Leal F, Del Caño-Ochoa F, Kirk EP, Ugarte M,Ramón-Maiques S, Gámez A, Pérez B. . Hum Mutat. 2022, 43(10):1430-1442.
Functional and structural deficiencies of Gemin5 variants associated with neurological disorders. Francisco-Velilla R, Embarc-Buh A, Del Caño-Ochoa F, Abellan S, Vilar M,Alvarez S, Fernandez-Jaen A, Kour S, Rajan DS, Pandey UB, Ramón-Maiques S,Martinez-Salas E. Life Sci Alliance. 2022, 5(7):e202201403.
Insight on molecular pathogenesis and pharmacochaperoning potential in phosphomannomutase 2 deficiency, provided by novel human phosphomannomutase 2 structures. Briso-Montiano A, Del Caño-Ochoa F, Vilas A, Velázquez-Campoy A, Rubio V, Pérez B, Ramón-Maiques S. J Inherit Metab Dis. 2022, Mar;45(2):318-333.
Deciphering CAD: Structure and function of a mega-enzymatic pyrimidine factory in health and disease. Del Caño-Ochoa F, Ramón-Maiques S. Protein Sci. 2021 30(10):1995-2008
Afatinib exerts immunomodulatory effects by targeting the pyrimidine biosynthesis enzyme CAD. Tu HF, Ko CJ, Lee CT, Lee CF, Lan SW, Lin HH, Lin HY, Ku CC, Lee DY, Chen IC, Chuang YH, Del Caño-Ochoa F, Ramón-Maiques S, Ho CC, Lee MS, Chang GD. Cancer Res. 2021 81(12):3270-3282
Mechanisms of feedback inhibition and sequential firing of active sites in plant aspartate transcarbamoylase. Bellin L, Del Caño-Ochoa F, Velázquez-Campoy A, Möhlmann T, Ramón-Maiques S. Nat Commun. 2021 12(1):947
The GATA3 X308_Splice breast cancer mutation is a hormone context-dependent oncogenic driver. Hruschka N, Kalisz M, Subijana M, Graña-Castro O, Del Cano-Ochoa F, Brunet LP, Chernukhin I, Sagrera A, De Reynies A, Kloesch B, Chin SF, Burgués O, Andreu D, Bermejo B, Cejalvo JM, Sutton J, Caldas C, Ramón-Maiques S, Carroll JS, Prat A, Real FX, Martinelli P. Oncogene 2020 39(32):5455-5467
Cell-based analysis of CAD variants identifies individuals likely to benefit from uridine therapy. Del Caño-Ochoa F, Ng BG, Abedalthagafi M, Almannai M, Cohn RD, Costain G, Elpeleg O, Houlden H, Karimiani EG, Liu P, Manzini MC, Maroofian R, Muriello M, Al-Otaibi A, Patel H, Shimon E, Sutton VR, Toosi MB, Wolfe LA, Rosenfeld JA, Freeze HH, Ramón-Maiques S. Genet Med. 2020 22(10):1598-1605
The multienzymatic protein CAD leading the de novo biosynthesis of pyrimidines localizes exclusively in the cytoplasm and does not translocate to the nucleus. Del Caño-Ochoa F, Ramón-Maiques S. Nucleosides Nucleotides Nucleic Acids. 2020 39(10-12):1320-1334
CAD, a multienzymatic protein at the head of de novo pyrimidine biosynthesis. Del Caño-Ochoa F, Moreno-Morcillo M, Ramón-Maiques S. Subcell Biochem. 2019 93:505-538
Structural basis for the dimerization of Gemin5 and its role in protein recruitment and translation control. Moreno-Morcillo M, Francisco-Velilla R, Embarc-Buh A, Fernández-Chamorro J, Ramón-Maiques S, Martinez-Salas E. Nucleic Acids Res. 2020 48(2):788-801
Characterization of the catalytic flexible loop in the dihydroorotase domain of the human multi-enzymatic protein CAD. Del Caño-Ochoa F, Grande-García A, Reverte-López M, D’Abramo M, Ramón-Maiques S. J Biol Chem. 2018 293(49):18903-18913
Gain-of-function mutations in DNMT3A in patients with paraganglioma. Remacha L, Currás-Freixes M, Torres-Ruiz R, Schiavi F, Torres-Pérez R, Calsina B, Letón R, Comino-Méndez I, Roldán-Romero JM, Montero-Conde C, Santos M, Pérez LI, Pita G, Alonso MR, Honrado E, Pedrinaci S, Crespo-Facorro B, Percesepe A, Falcioni M, Rodríguez-Perales S, Korpershoek E, Ramón-Maiques S, Opocher G, Rodríguez-Antona C, Robledo M, Cascón A. Genet Med. 2018 20(12):1644-1651
CAD: A multifunctional protein leading de novo pyrimidine biosynthesis. Moreno-Morcillo M, Ramón-Maiques S. Encyclopedia of Life Sciences (eLS) 2017. John Wiley & Sons, Ltd: Chichester. DOI: 10.1002/9780470015902.a0027193
Structural insight into the core of CAD, the multifunctional protein leading de novo pyrimidine biosynthesis. Moreno-Morcillo M, Grande-García A, Ruiz-Ramos A, Del Caño-Ochoa F, Boskovic J, Ramón-Maiques S. Structure. 2017 25(6):912-923.e5
Structure and functional characterization of human aspartate transcarbamoylase, the target of the anti-tumoral drug PALA. Ruiz-Ramos A, Velázquez-Campoy A, Grande-García A, Moreno-Morcillo M, Ramón-Maiques S. Structure. 2016 24(7):1081-94
The N-terminal domain of MuB protein has striking structural similarity to DNA-binding domains and mediates MuB filament-filament interactions. Dramićanin M, López-Méndez B, Boskovic J, Campos-Olivas R, Ramón-Maiques S. J Struct Biol. 2015 191(2):100-11.
MuB gives a new twist to target DNA selection. Dramićanin M, Ramón-Maiques S. Mob Genet Elements. 2013, 3(5):e27515.
Structure, functional characterization, and evolution of the dihydroorotase domain of human CAD. Grande-García A, Lallous N, Díaz-Tejada C, Ramón-Maiques S. Structure. 2014, 22(2):185-98.
Expression, purification, crystallization and preliminary X-ray diffraction analysis of the aspartate transcarbamoylase domain of human CAD. Ruiz-Ramos A, Lallous N, Grande-García A, Ramón-Maiques S. Acta Crystallogr Sect F Struct Biol Cryst Commun. 2013, 69(Pt 12):1425-30.
MuB is an AAA+ ATPase that forms helical filaments to control target selection for DNA transposition. Mizuno N, Dramićanin M, Mizuuchi M, Adam J, Wang Y, Han YW, Yang W, Steven AC, Mizuuchi K, Ramón-Maiques S. Proc Natl Acad Sci USA. 2013, 110(27):E2441-50.
Expression, purification, crystallization and preliminary X-ray diffraction analysis of the dihydroorotase domain of human CAD. Lallous N, Grande-García A, Molina R, Ramón-Maiques S. Acta Crystallogr Sect F Struct Biol Cryst Commun. 2012, 68(Pt 11):1341-5.
The PHD finger of human UHRF1 reveals a new subgroup of unmethylated histone H3 tail readers. Lallous N, Legrand P, McEwen AG, Ramón-Maiques S, Samama JP, Birck C. PLoS One. 2011;6(11):e27599.
We are happy and honored to have had many awesome people in our lab.
Thank you all for sharing your passion for Science and your friendship.
Francisco (Curro) Vicente (Spain), Marta Fernández (Spain), Eliska Smirakova (Czech Republic), Daniela Caijao (Colombia), Luisa Gleich (Germany), Patricia Exposito (Spain), Manuel Garavito (Colombia), Fernando Valenzuela (Spain), Leo Bellin (Germany), Alessio Falzone (Germany), Uxía Pérez (Spain), Laura García (Spain), Rebeca Company (Spain), Aneliya Dragomirova (Spain), Miriam Poley (Spain), Vanessa Scherer (Germany), Alejandro Alarcón (Austria), Lobna Ramadane (Spain), Juan Luis Gallego (Spain), Jordi Juan Girnoés (Spain), Jose Antonio Ferrandis (Spain), Pablo López (Spain)