Ernesto Arias-Palomo

Structure of Epac2 in complex with a cyclic AMP analogue and RAP1B

Epac proteins are activated by binding of the second messenger cAMP and then act as guanine nucleotide exchange factors for Rap proteins. The Epac proteins are involved in the regulation of cell adhesion and insulin secretion. Here we have determined the structure of Epac2 in complex with a cAMP analogue (Sp-cAMPS) and RAP1B by X-ray crystallography and single particle electron microscopy. The structure represents the cAMP activated state of the Epac2 protein with the RAP1B protein trapped in the course of the exchange reaction. Comparison with the inactive conformation reveals that cAMP binding causes conformational changes that allow the cyclic nucleotide binding domain to swing from a position blocking the Rap binding site towards a docking site at the Ras exchange motif domain.

Architecture of the Pontin/Reptin Complex, Essential in the Assembly of Several Macromolecular Complexes

Pontin and reptin belong to the AAA+ family, and they are essential for the structural integrity and catalytic activity of several chromatin remodeling complexes. They are also indispensable for the assembly of several ribonucleoprotein complexes, including telomerase. Here, we propose a structural model of the yeast pontin/reptin complex based on a cryo-electron microscopy reconstruction at 13 A. Pontin/reptin hetero-dodecamers were purified from in vivo assembled complexes forming a double ring. Two rings interact through flexible domains projecting from each hexamer, constituting an atypical asymmetric form of oligomerization. These flexible domains and the AAA+ cores reveal significant conformational changes when compared with the crystal structure of human pontin that generate enlarged channels. This structure of endogenously assembled pontin/reptin complexes is different than previously described structures, suggesting that pontin and reptin could acquire distinct structural states to regulate their broad functions as molecular motors and scaffolds for nucleic acids and proteins.

The nonsense-mediated mRNA decay SMG-1 kinase is regulated by large-scale conformational changes controlled by SMG-8

Nonsense-mediated mRNA decay (NMD) is a eukaryotic surveillance pathway that regulates the degradation of mRNAs harboring premature translation termination codons. NMD also influences the expression of many physiological transcripts. SMG-1 is a large kinase essential to NMD that phosphorylates Upf1, which seems to be the definitive signal triggering mRNA decay. However, the regulation of the kinase activity of SMG-1 remains poorly understood. Here, we reveal the three-dimensional architecture of SMG-1 in complex with SMG-8 and SMG-9, and the structural mechanisms regulating SMG-1 kinase. A bent arm comprising a long region of HEAT (huntington, elongation factor 3, a subunit of PP2A and TOR1) repeats at the N terminus of SMG-1 functions as a scaffold for SMG-8 and SMG-9, and projects from the C-terminal core containing the phosphatidylinositol 3-kinase domain. SMG-9 seems to control the activity of SMG-1 indirectly through the recruitment of SMG-8 to the N-terminal HEAT repeat region of SMG-1. Notably, SMG-8 binding to the SMG-1:SMG-9 complex specifically down-regulates the kinase activity of SMG-1 on Upf1 without contacting the catalytic domain. Assembly of the SMG-1:SMG-8:SMG-9 complex induces a significant motion of the HEAT repeats that is signaled to the kinase domain. Thus, large-scale conformational changes induced by SMG-8 after SMG-9-mediated recruitment tune SMG-1 kinase activity to modulate NMD.

Global conformational rearrangements during the activation of the GDP/GTP exchange factor Vav3

Activation of Rho/Rac GTPases during cell signaling requires the participation of GDP/GTP exchange factors of the Dbl family. Although the structure of the catalytic core of Dbl proteins has been established recently, the molecular changes that the full‐length proteins experience during normal or oncogenic conditions of stimulation are still unknown. Here, we have used single‐particle electron microscopy to solve the structures of the inactive (unphosphorylated), active (phosphorylated), and constitutively active (N‐terminally deleted) versions of the exchange factor Vav3. Comparison of these forms has revealed the interdomain interactions maintaining the inactive Vav3 state and the dynamic changes that the overall Vav3 structure undergoes upon tyrosine phosphorylation. We have also found that the conformations of phosphorylated Vav3 and N‐terminally deleted Vav3 are distinct, indicating that the acquisition of constitutive activity by exchange factors is structurally more complex than the mere elimination of inhibitory interactions between structural domains.

Characterization of SMG-9, an essential component of the nonsense-mediated mRNA decay SMG1C complex

SMG-9 is part of a protein kinase complex, SMG1C, which consists of the SMG-1 kinase, SMG-8 and SMG-9. SMG1C mediated phosphorylation of Upf1 triggers nonsense-mediated mRNA decay (NMD), a eukaryotic surveillance pathway that detects and targets for degradation mRNAs harboring premature translation termination codons. Here, we have characterized SMG-9, showing that it comprises an N-terminal 180 residue intrinsically disordered region (IDR) followed by a well-folded C-terminal domain. Both domains are required for SMG-1 binding and the integrity of the SMG1C complex, whereas the C-terminus is sufficient to interact with SMG-8. In addition, we have found that SMG-9 assembles in vivo into SMG-9:SMG-9 and, most likely, SMG-8:SMG-9 complexes that are not constituents of SMG1C. SMG-9 self-association is driven by interactions between the C-terminal domains and surprisingly, some SMG-9 oligomers are completely devoid of SMG-1 and SMG-8. We propose that SMG-9 has biological functions beyond SMG1C, as part of distinct SMG-9-containing complexes. Some of these complexes may function as intermediates potentially regulating SMG1C assembly, tuning the activity of SMG-1 with the NMD machinery. The structural malleability of IDRs could facilitate the transit of SMG-9 through several macromolecular complexes.

Electron microscopy of Xrcc4 and the DNA ligase IV-Xrcc4 DNA repair complex.

The DNA ligase IV-Xrcc4 complex is responsible for the ligation of broken DNA ends in the non-homologous end-joining (NHEJ) pathway of DNA double strand break repair in mammals. Mutations in DNA ligase IV (Lig4) lead to immunodeficiency and radiosensitivity in humans. Only partial structural information for Lig4 and Xrcc4 is available, while the structure of the full-length proteins and their arrangement within the Lig4-Xrcc4 complex is unknown. The C-terminal domain of Xrcc4, whose structure has not been solved, contains phosphorylation sites for DNA-PKcs and is phylogenetically conserved, indicative of a regulatory role in NHEJ. Here, we have purified full length Xrcc4 and the Lig4-Xrcc4 complex, and analysed their structure by single-particle electron microscopy. The three-dimensional structure of Xrcc4 at a resolution of approximately 37A reveals that the C-terminus of Xrcc4 forms a dimeric globular domain connected to the N-terminus by a coiled-coil. The N- and C-terminal domains of Xrcc4 locate at opposite ends of an elongated molecule. The electron microscopy images of the Lig4-Xrcc4 complex were examined by two-dimensional image processing and a double-labelling strategy, identifying the site of the C-terminus of Xrcc4 and the catalytic core of Lig4 within the complex. The catalytic domains of Lig4 were found to be in the vicinity of the N-terminus of Xrcc4. We provide a first sight of the structural organization of the Lig4-Xrcc4 complex, which suggests that the BRCT domains could provide the link of the ligase to Xrcc4 while permitting some movements of the catalytic domains of Lig4. This arrangement may facilitate the ligation of diverse configurations of damaged DNA.

Biochemical Characterization of the Transcriptional Regulator BzdR from Azoarcus sp. CIB

The BzdR transcriptional regulator that controls the PN promoter responsible for the anaerobic catabolism of benzoate in Azoarcus sp. CIB constitutes the prototype of a new subfamily of transcriptional regulators. Here, we provide some insights about the functional-structural relationships of the BzdR protein. Analytical ultracentrifugation studies revealed that BzdR is homodimeric in solution. An electron microscopy three-dimensional reconstruction of the BzdR dimer has been obtained, and the predicted structures of the respective N- and C-terminal domains of each BzdR monomer could be fitted into such a reconstruction. Gel retardation and ultracentrifugation experiments have shown that the binding of BzdR to its cognate promoter is cooperative. Different biochemical approaches revealed that the effector molecule benzoyl-CoA induces conformational changes in BzdR without affecting its oligomeric state. The BzdR-dependent inhibition of the PN promoter and its activation in the presence of benzoyl-CoA have been established by in vitro transcription assays. The monomeric BzdR4 and BzdR5 mutant regulators revealed that dimerization of BzdR is essential for DNA binding. Remarkably, a BzdRΔL protein lacking the linker region connecting the N- and C-terminal domains of BzdR is also dimeric and behaves as a super-repressor of the PN promoter. These data suggest that the linker region of BzdR is not essential for protein dimerization, but rather it is required to transfer the conformational changes induced by the benzoyl-CoA to the DNA binding domain leading to the release of the repressor. A model of action of the BzdR regulator has been proposed.

Conformational rearrangements upon Syk auto-phosphorylation

Syk is a cytoplasmic tyrosine kinase that is activated after recruitment to immune receptors, triggering the phopshorylation of downstream targets. The kinase activity of Syk is controlled by an auto-inhibited conformation consisting of a regulatory region that contains two N-terminal Src homology 2 (SH2) domains inhibiting the catalytic activity of the kinase domain located at the C-terminus. The atomic structure of the related Zap-70 kinase and an electron microscopy (EM) model of Syk have revealed the structural mechanism of this auto-inhibition based on the formation of a compact conformation sustained by interactions between the regulatory and catalytic domains. On the other hand, the structural basis of Syk activation is not fully understood due to the lack of a 3D structure of full-length Syk in an active conformation. Here, we have used single-particle electron microscopy to analyse the conformational changes taking place in an activated form of Syk induced by auto-phosphorylation. The conformation of phosphorylated Syk is reminiscent of the compact structure of the inhibited protein but significant conformational changes are observed in the regulatory region. These rearrangements could be sufficient to disrupt the inhibitory interactions, contributing to Syk activation. These results suggest that the regulation of the activation of Syk might be modulated by subtle changes in the positioning of the regulatory domains rather than a full opening mechanism as proposed for the Src kinases.

A new protein carrying an NmrA-like domain is required for cell differentiation and development in Dictyostelium discoideum

We have isolated a Dictyostelium mutant unable to induce expression of the prestalk-specific marker ecmB in monolayer assays. The disrupted gene, padA, leads to a range of phenotypic defects in growth and development. We show that padA is essential for growth, and we have generated a thermosensitive mutant allele, padA(-). At the permissive temperature, mutant cells grow poorly; they remain longer at the slug stage during development and are defective in terminal differentiation. At the restrictive temperature, growth is completely blocked, while development is permanently arrested prior to culmination. padA(-) slugs are deficient in prestalk A cell differentiation and present an abnormal ecmB expression pattern. Sequence comparisons and predicted three-dimensional structure analyses show that PadA carries an NmrA-like domain. NmrA is a negative transcriptional regulator involved in nitrogen metabolite repression in Aspergillus nidulans. PadA predicted structure shows a NAD(P)(+)-binding domain, which we demonstrate that is essential for function. We show that padA(-) development is more sensitive to ammonia than wild-type cells and two ammonium transporters, amtA and amtC, appear derepressed during padA(-) development. Our data suggest that PadA belongs to a new family of NAD(P)(+)-binding proteins that link metabolic changes to gene expression and is required for growth and normal development.