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10 páginas, 7 figuras, Gemin5 is a multifunctional RNA binding protein (RBP) organized in domains with a distinctive structural organization. The protein is a hub for several protein networks performing diverse RNA-dependent functions including regulation of translation, and recognition of small nuclear RNAs (snRNAs). Here we sought to identify the presence of phosphoresidues on the C-terminal half of Gemin5, a region of the protein that harbors a tetratricopeptide repeat (TPR)-like dimerization domain and a non-canonical RNA binding site (RBS1). We identified two phosphoresidues in the purified protein: P-T897 in the dimerization domain and P-T1355 in RBS1. Replacing T897 and T1355 with alanine led to decreased translation, and mass spectrometry analysis revealed that mutation T897A strongly abrogates the association with cellular proteins related to the regulation of translation. In contrast, the phosphomimetic substitutions to glutamate partially rescued the translation regulatory activity. The structural analysis of the TPR dimerization domain indicates that local rearrangements caused by phosphorylation of T897 affect the conformation of the flexible loop 2-3, and propagate across the dimerization interface, impacting the position of the C-terminal helices and the loop 12-13 shown to be mutated in patients with neurological disorders. Computational analysis of the potential relationship between post-translation modifications and currently known pathogenic variants indicates a lack of overlapping of the affected residues within the functional domains of the protein and provides molecular insights for the implication of the phosphorylated residues in translation regulation., This work was supported by the Ministerio de Ciencia e Innovación (MICIN) and Fondo Europeo de Desarrollo Regional (AEI/FEDER UE) (PID2020-115096RB-I00), Comunidad de Madrid (B2017/BMD-3770) and an Institutional grant from Fundación Ramón Areces. FdC is a postdoctoral fellow of the Generalitat Valenciana (APOSTD 2021).

MuB is an ATP-dependent DNA-binding protein that regulates the activity of MuA transposase and delivers the target DNA for transposition of phage Mu. Mechanistic insight into MuB function is limited to its AAA+ ATPase module, which upon ATP binding assembles into helical filaments around the DNA. However, the structure and function of the flexible N-terminal domain (NTD) appended to the AAA+ module remains uncharacterized. Here we report the solution structure of MuB NTD determined by NMR spectroscopy. The structure reveals a compact domain formed by four α-helices connected by short loops, and confirms the presence of a helix-turn-helix motif. High structural similarity and sequence homology with λ repressor-like DNA-binding domains suggest a possible role of MuB NTD in DNA binding. We also demonstrate that the NTD directly mediates the ability of MuB to establish filament-filament interactions. These findings lead us to a model in which the NTD interacts with the AAA+ spirals and perhaps also with the DNA bound within the filament, favoring MuB polymerization and filament clustering. We propose that the MuB NTD-dependent filament interactions might be an effective mechanism to bridge distant DNA regions during Mu transposition.

SummaryUpregulation of CAD, the multifunctional protein that initiates and controls the de novo biosynthesis of pyrimidines in animals, is essential for cell proliferation. Deciphering the architecture and functioning of CAD is of interest for its potential usage as an antitumoral target. However, there is no detailed structural information about CAD other than that it self-assembles into hexamers of ∼1.5 MDa. Here we report the crystal structure and functional characterization of the dihydroorotase domain of human CAD. Contradicting all assumptions, the structure reveals an active site enclosed by a flexible loop with two Zn2+ ions bridged by a carboxylated lysine and a third Zn coordinating a rare histidinate ion. Site-directed mutagenesis and functional assays prove the involvement of the Zn and flexible loop in catalysis. Comparison with homologous bacterial enzymes supports a reclassification of the DHOase family and provides strong evidence against current models of the architecture of CAD.

Carbamate kinase (CK) makes ATP from ADP and carbamoyl phosphate (CP) in the final step of the microbial fermentative catabolism of arginine, agmatine, and oxalurate/allantoin. Two previously reported CK structures failed to clarify CP binding and catalysis and to reveal the significance of the protruding subdomain (PSD) that hangs over the CK active center as an exclusive and characteristic CK feature. We clarify now these three questions by determining two crystal structures of Enterococcus faecalis CK (one at 1.5 A resolution and containing bound MgADP, and the other at 2.1 A resolution and having in the active center one sulfate and two fixed water molecules that mimic one bound CP molecule) and by mutating active-center residues, determining the consequences of these mutations on enzyme functionality. Superimposition of the present crystal structures reconstructs the filled active center in the ternary complex, immediately suggesting in-line associative phosphoryl group transfer and a mechanism for enzyme catalysis involving N51, K209, K271, D210, and the PSD residue K128. The large respective increases and decreases in K(m)(CP) and k(cat) triggered by the mutations N51A, K128A, K209A, and D210N corroborate the ternary complex active-site architecture and the catalytic mechanism proposed. The extreme negative effects of K128A demonstrate a key role of the PSD in substrate binding and catalysis. The crystal structures reveal large rigid-body movements of the PSD towards the enzyme body that place K128 next to CP and bury the CP site. A mechanism that connects CP site occupation with the PSD approach, involving V206-I207 in the CP site and P162-S163 in the PSD stem, is identified. The effects of the V206A and V206L mutations support this mechanism. It is concluded that the PSD movement allows CK to select against the abundant CP/carbamate analogues acetylphosphate/acetate and bicarbonate, rendering CK highly selective for CP/carbamate., This work was supported by grant BFU2008-05021 from the Spanish Ministry for Science and grant Prometeo/2009/051 from the Valencian Government. S.R.-M. and F.G.-O. are postdoctoral fellows (JAE-Doc) of the Consejo Superior de Investigaciones Científicas. A.G. was a recipient of a Consejo Superior de Investigaciones Científicas fellowship for undergraduate students (JAE-Intro).

We test, using site-directed mutagenesis, predictions based on the X-ray structure of N-acetyl-L-glutamate kinase (NAGK), the paradigm of the amino acid kinase protein family, about the roles of specific residues on substrate binding and catalysis. The mutations K8R and D162E decreased V([sustrate]= infinity ) 100-fold and 1000-fold, respectively, in agreement with the predictions that K8 catalyzes phosphoryl transfer and D162 organizes the catalytic groups. R66K and N158Q increased selectively K(m)(Asp) three to four orders of magnitude, in agreement with the binding of R66 and N158 to the C(alpha) substituents of NAG. Mutagenesis in parallel of aspartokinase III (AKIII phosphorylates aspartate instead of acetylglutamate), another important amino acid kinase family member of unknown 3-D structure, identified in AKIII two residues, K8 and D202, that appear to play roles similar to those of K8 and D162 of NAGK, and supports the involvement of E119 and R198, similarly to R66 and N158 of NAGK, in the binding of the amino acid substrate, apparently interacting, respectively, with the alpha-NH(3)(+) and alpha-COO(-) of aspartate. These results and an improved alignment of the NAGK and AKIII sequences have guided us into 3-D modelling of the amino acid kinase domain of AKIII using NAGK as template. The model has good stereochemistry and validation parameters. It provides insight into substrate binding and catalysis, agreeing with mutagenesis results with another aspartokinase that were not considered when building the model.AKIII is homodimeric and is inhibited by lysine. Lysine may bind to a regulatory region that is C-terminal to the amino acid kinase domain. We make a C-terminally truncated AKIII (AKIIIt) and show that the C-region is involved in intersubunit interactions, since AKIIIt is found to be monomeric. Further, it is inactive, as demanded if dimer formation is essential for activity. Models for AKIII architecture are proposed that account for these findings.

CAD, the multifunctional protein initiating and controlling de novo biosynthesis of pyrimidines in animals, self-assembles into ∼1.5 MDa hexamers. The structures of the dihydroorotase (DHO) and aspartate transcarbamoylase (ATC) domains of human CAD have been previously determined, but we lack information on how these domains associate and interact with the rest of CAD forming a multienzymatic unit. Here, we prove that a construct covering human DHO and ATC oligomerizes as a dimer of trimers and that this arrangement is conserved in CAD-like from fungi, which holds an inactive DHO-like domain. The crystal structures of the ATC trimer and DHO-like dimer from the fungus Chaetomium thermophilum confirm the similarity with the human CAD homologs. These results demonstrate that, despite being inactive, the fungal DHO-like domain has a conserved structural function. We propose a model that sets the DHO and ATC complex as the central element in the architecture of CAD., Spanish Ministry of Economy and Competitiveness (MINECO; BFU2013-48365-P and BFU2016-80570-R)

Carbamoyl phosphate (CP), the essential precursor of pyrimidines and arginine, is made in Escherichia coli by a single carbamoyl phosphate synthetase (CPS) consisting of 41.4 and 117.7 kDa subunits, which is feed-back inhibited by UMP and activated by IMP and ornithine. The large subunit catalyzes CP synthesis from ammonia in three steps, and binds the effectors in its 15 kDa C-terminal domain. Fifteen site-directed mutations were introduced in 13 residues of this domain to investigate the mechanism of allosteric modulation by UMP and IMP. Two mutations, K993A and V994A, decreased significantly or abolished enzyme activity, apparently by interfering with the step of carbamate synthesis, and one mutation, T974A, negatively affected ornithine activation. S948A, K954A, T974A, K993A and K993W/H995A abolished or greatly hampered IMP activation and UMP inhibition as well as the binding of both effectors, monitored using photoaffinity labeling and ultracentrifugation binding assays. V994A also decreased significantly IMP and UMP binding. L990A, V991A, H995A, G997A and G1008A had more modest effects or affected more the modulation by and the binding of one than of the other nucleotide. K993W, R1020A, R1021A and K1061A were without substantial effects. The results confirm the independence of the regulatory and catalytic centers, and also confirm functional predictions based on the X-ray structure of an IMP-CPS complex. They prove that the inhibitor UMP and the activator IMP bind in the same site, and exclude that the previously observed binding of ornithine and glutamine in this site were relevant for enzyme activation. K993 and V994 appear to be involved in the transmission of the regulatory signals triggered by UMP and IMP binding. These effectors possibly change the position of K993 and V994, and alter the intermolecular contacts mediated by the regulatory domain.

Carbamoyl phosphate (CP), an essential precursor of arginine and the pyrirnidine bases, is synthesized by CP synthetase (CPS) in three steps. The last step, the phosphorylation of carbamate, is also catalyzed by carbamate kinase (CK), an enzyme used by microorganisms to produce ATP from ADP and CP. Although the recently determined structures of CPS and CK show no obvious mutual similarities, a CK-like CPS reported in hyperthermophilic archaea was postulated to be a missing link in the evolution of CP biosynthesis. The 1.5 Å resolution structure of this enzyme from Pyrococcus furiosus shows both a subunit topology and a homodimeric molecular organization, with a 16-stranded open β-sheet core surrounded by α-helices, similar to those in CK. However, the pyrococcal enzyme exhibits many solvent-accessible ion-pairs, an extensive, strongly hydrophobic, intersubunit surface, and presents a bound ADP molecule, which does not dissociate at 22°C from the enzyme. The ADP nucleotide is sequestered in a ridge formed over. the C-edge of the core sheet, at the bottom of a large cavity, with the purine ring enclosed in a pocket specific for adenine. Overall, the enzyme structure is ill-suited for catalyzing the characteristic three-step reaction of CPS and supports the view that the CK-like CPS is in fact a highly thermostable and very slow (at 37°C) CK that, in the extreme environment of P. furiosus, may have the new function of making, rather than using, CP. The thermostability of the enzyme may result from the extension of the hydrophobic intersubunit contacts and from the large number of exposed ion-pairs, some of which form ion-pair networks across several secondary structure elements in each enzyme subunit. The structure provides the first information on substrate binding and catalysis in CKs, and suggests that the slow rate at 37°C is possibly a consequence of slow product dissociation. (C) 2000 Academic Press., We thank the EU, ESRF and EMBL Grenoble for financial assistance and support for data collection. This work was supported by grants PM97-0134-C02-01 and PB95–0218 of the Dirección General de Enseñ anza Superior (DGES) of Spain. S.R.-M. is a predoctoral fellow of the Generalitat Valenciana and M.U. was a postdoctoral fellow of the Fundación Valenciana de Investigaciones Biomédicas-Bancaixa

V(D)J recombination is central to establishing a functional adaptive immune system. The large repertoire of immunoglobulins and T-cell receptors is generated by combinatorial rearrangement of an extensive array of variable (V), diversity (D), and joining (J) gene segments that are joined to encode the variable domains of the protein chains. The recombination signal sequences (RSS) that flank these gene segments are recognized, paired in a synaptic complex, and cleaved by collaboration of the lymphoid-specific proteins RAG1 and RAG2. After cleavage, the signal ends remain tightly bound to the RAG proteins in a particularly stable Signal-End Complex (SEC).To obtain 3D structural information about RAG1/2 bound to RSS DNA, isolated and purified SEC were visualized by AFM. To better define the arrangement of the RAG proteins and RSS DNA in the complex, we used RAG1 and RAG2 fused with maltose binding protein (MBP). A wide variety of complex shapes was recorded, however, it was clear that the two DNA chains predominantly exited the SEC complex from adjacent points. The volume of the protein core was consistent with the expected mass of 500 kDa corresponding to (RAG1)2-(RAG2)2 composition. MBP protrusions could be observed on the protein particles marking the N-termini of RAG1 and RAG2. To make their appearance more noticeable, we used selective antibody labeling. Fab-labeled MBPs were clearly identified peripheral to the recombinase core. When only the RAG2 MBPs were labeled, the two DNAs most often exited together from the SEC on the opposite side to the Fabs. Consistent with this observation, when only the RAG1 MBPs were labeled, they were situated closer to the exiting DNAs.The parallel arrangement of DNA and protein subunits found by AFM is in an excellent agreement with the 3D model based on EM data.

The publisher regrets that Ref. 32 was incorrectly cited. For the reader's convenience, the correctcitation appears here:Marcos, E., Crehuet, R. & Bahar, I. (2010). On the conservation of the slow conformational dynamicswithin the amino acid kinase family: NAGK the paradigm. PLoS Comput. Biol., 6, e1000738.