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Acta Crystallographica Section D: Biological Crystallography (1)
Journal of Bacteriology (1)
Journal of molecular biology (1)
Vonrhein, Clemens (3)
Bricogne, Gérard (2)
Bricogne, Gerard (1)
Chen, Ming Wei (1)
Chuah, Mary Lay Cheng (1)
Flensburg, Claus (1)
Izard, Tina (1)
Keller, Peter (1)
Kotaka, Masayo (1)
Lee, Jun Hyuck (1)
Lescar, Julien (1)
Liang, Zhao-Xun (1)
Paciorek, Włodek (1)
Rangarajan, Erumbi S. (1)
Rao, Feng (1)
Schneider, Gunter (1)
Sharff, Andrew (1)
Smart, Oliver S. (1)
Svergun, Dmitri (1)
Womack, Thomas O. (1)
Year of Publication
The metavinculin tail domain directs constitutive interactions with raver1 and vinculin RNA
Lee, Jun Hyuck
Rangarajan, Erumbi S.
Journal of molecular biology
Vinculin is a key regulator of the attachment of the actin cytoskeleton to the cell membrane at cellular adhesion sites that is crucial for processes like cell motility and migration, development, survival, and wound healing. Vinculin loss results in embryonic lethality, cardiovascular diseases, and cancer. Its tail domain, Vt, is crucial for vinculin activation and focal adhesion turnover and binds to the actin cytoskeleton and acidic phospholipids upon which it unfurls. The RNA binding protein raver1 regulates the assembly of focal adhesions transcriptionally by binding to vinculin. The muscle-specific splice form, metavinculin, is characterized by a 68 residue insert in the tail domain (MVt) and correlates with hereditary idiopathic dilated cardiomyopathy. Here we report that metavinculin can bind to raver1 in its inactive state. Our crystal structure explains this permissivity, where an extended coil unique to MVt is unfurled in the MVtΔ954:raver1 complex structure. Our binding assays show that raver1 forms a ternary complex with MVt and vinculin mRNA. These findings suggest that the metavinculin:raver1:RNA complex is constitutively recruited to adhesion complexes.
adherens junction; cardiomyopathy; focal adhesion; RRM domain; RNA binding
Structural Insights into the Regulatory Mechanism of the Response Regulator RocR from Pseudomonas aeruginosa in Cyclic Di-GMP Signaling
Chen, Ming Wei
Chuah, Mary Lay Cheng
Journal of Bacteriology
The nucleotide messenger cyclic di-GMP (c-di-GMP) plays a central role in the regulation of motility, virulence, and biofilm formation in many pathogenic bacteria. EAL domain-containing phosphodiesterases are the major signaling proteins responsible for the degradation of c-di-GMP and maintenance of its cellular level. We determined the crystal structure of a single mutant (R286W) of the response regulator RocR from Pseudomonas aeruginosa to show that RocR exhibits a highly unusual tetrameric structure arranged around a single dyad, with the four subunits adopting two distinctly different conformations. Subunits A and B adopt a conformation with the REC domain located above the c-di-GMP binding pocket, whereas subunits C and D adopt an open conformation with the REC domain swung to the side of the EAL domain. Remarkably, the access to the substrate-binding pockets of the EAL domains of the open subunits C and D are blocked in trans by the REC domains of subunits A and B, indicating that only two of the four active sites are engaged in the degradation of c-di-GMP. In conjunction with biochemical and biophysical data, we propose that the structural changes within the REC domains triggered by the phosphorylation are transmitted to the EAL domain active sites through a pathway that traverses the dimerization interfaces composed of a conserved regulatory loop and the neighboring motifs. This exquisite mechanism reinforces the crucial role of the regulatory loop and suggests that similar regulatory mechanisms may be operational in many EAL domain proteins, considering the preservation of the dimerization interface and the spatial arrangement of the regulatory domains.
Exploiting structure similarity in refinement: automated NCS and target-structure restraints in BUSTER
Smart, Oliver S.
Womack, Thomas O.
Acta Crystallographica Section D: Biological Crystallography
Local structural similarity restraints (LSSR) provide a novel method for exploiting NCS or structural similarity to an external target structure. Two examples are given where BUSTER re-refinement of PDB entries with LSSR produces marked improvements, enabling further structural features to be modelled.
Maximum-likelihood X-ray macromolecular structure refinement in BUSTER has been extended with restraints facilitating the exploitation of structural similarity. The similarity can be between two or more chains within the structure being refined, thus favouring NCS, or to a distinct ‘target’ structure that remains fixed during refinement. The local structural similarity restraints (LSSR) approach considers all distances less than 5.5 Å between pairs of atoms in the chain to be restrained. For each, the difference from the distance between the corresponding atoms in the related chain is found. LSSR applies a restraint penalty on each difference. A functional form that reaches a plateau for large differences is used to avoid the restraints distorting parts of the structure that are not similar. Because LSSR are local, there is no need to separate out domains. Some restraint pruning is still necessary, but this has been automated. LSSR have been available to academic users of BUSTER since 2009 with the easy-to-use -autoncs and -target target.pdb options. The use of LSSR is illustrated in the re-refinement of PDB entries 5rnt, where -target enables the correct ligand-binding structure to be found, and 1osg, where -autoncs contributes to the location of an additional copy of the cyclic peptide ligand.
BUSTER; NCS restraints; target-structure restraints; local structural similarity restraints
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