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Title

Crystal structures of human carbamoyl phosphate synthetase 1 (CPS1) shed light on domains functions, substrate tunnels and allosteric activation, and allow rationalization of inborn CPS1 deficiency

AuthorsCima, Sergio de; Polo, Luis Mariano ; Díez-Fernández, Carmen ; Cervera, Javier; Fita, Ignacio ; Rubio, Vicente
Issue Date11-Sep-2014
PublisherSociedad Española de Bioquímica y Biología Molecular
CitationXXXVII Congreso de la Sociedad Española de Bioquímica y Biología Molecular (2014)
AbstractABSTRACT: P10-53 CPS1 is a large six-domain protein that constitutes the first step of the urea cycle: the synthesis of carbamoyl phosphate from bicarbonate, ammonia and two ATP molecules. A paramount feature of this enzyme is its absolute requirement for N-acetyl-L-glutamate (NAG), an allosteric activator without which it is inactive. The report of CPS1 regulation by lysine deacylation by NAD-dependent sirtuin 5 connected the urea cycle with the age-control machinery (Nakagawa et al. Cell 2009; 137:560). CPS1 deficiency (CPS1D) is an inborn disorder that cause severe neonatal hyperammonemia leading to mental retardation or even to death. More than 300 mutations have been reported in CPS1D patients, of which the majority are missense mutations showing little recurrence and having unproven disease-causing potential. The structure of the E. coli homologous CPS has been known for >15 years, but differences with CPS1 (40% identity; use of glutamine instead of ammonia; insensitivity to NAG) rendered essential to obtain the structure of CPS1 for proper understanding of its functioning, and for evaluating disease causation by CPS1D mutations. Using a baculovirus/insect cell system we have finally succeeded in producing recombinant human CPS1 in large amount and pure form, allowing us to experimentally examine the effects of reported mutations and ascertain its disease-causing potential (Díez-Fernández et al. Human Mut 2013; 34:1149). In addition we have determined the structure of CPS1, in both apo and ligand-bound (NAG and ADP/Pi) forms. The liganded structure revealed how NAG binds in a pocket of the C-terminal domain and has identified elements stabilized by ADP binding and conformational changes that lead to define the carbamate tunnel, which in the apo form is heavily branched and open to the environment. Our structures decipher the CPS1 inability to use glutamine and reveal a potential channel for ammonia intake. Furthermore, they help rationalize the disease-causing role of most clinical CPS1 mutations. Supported by Fundación Alicia Koplowitz and Valencian (Prometeo 2009/051) and Spanish (BFU2011-30407; FPU to CD-F) governments.
DescriptionComunicación presentada en el XXXVII Congreso de la Sociedad Española de Bioquímica y Biología Molecular SEBBM Granada2014, celebrada del 9 al 12 de septiembre de 2014 en Granada (España)
URIhttp://hdl.handle.net/10261/111093
Appears in Collections:(IBMB) Comunicaciones congresos
(IBV) Comunicaciones congresos
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