Interactions and structural variability of ß-carboxysomal shell protein CcmL.

CcmL is a small, pentameric protein that is argued to fill the vertices of ß-carboxysomal shell. Here we report the structures of two CcmL orthologs, those from Nostoc sp. PCC 7120 and Thermosynechococcus elongatus BP-1. These structures broadly resemble those previously reported for other strains. However, the Nostoc CcmL structure shows an interesting pattern of behavior where two loops that map to the base of the pentamer adopt either an out or in conformation, with a consistent (over six pentamers) out-in-out-in-in pattern of protomers. The pentamers in this structure are also consistently organized into a back-to-back decamer, though evidence suggests that this is likely not present in solution. Förster resonance energy transfer experiments were able to show a weak interaction between CcmL and CcmK2 when CcmK2 was present at >100 µM. Since CcmK2 forms defined bodies with approximately 200 nm diameter at this concentration, this would support the idea that CcmL can only interact with CcmK2 at rare defect points in the growing shell.

Subdomain location of mutations in cardiac actin correlate with type of functional change.

Determining the molecular mechanisms that lead to the development of heart failure will help us gain better insight into the most costly health problem in the Western world. To understand the roles that the actin protein plays in the development of heart failure, we have taken a systematic approach toward characterizing human cardiac actin mutants that have been associated with either hypertrophic or dilated cardiomyopathy. Seven known cardiac actin mutants were expressed in a baculovirus system, and their intrinsic properties were studied. In general, the changes to the properties of the actin proteins themselves were subtle. The R312H variant exhibited reduced stability, with a T(m) of 53.6 °C compared to 56.8 °C for WT actin, accompanied with increased polymerization critical concentration and Pi release rate, and a marked increase in nucleotide release rates. Substitution of methionine for leucine at amino acid 305 showed no impact on the stability, nucleotide release rates, or DNase-I inhibition ability of the actin monomer; however, during polymerization, a 2-fold increase in Pi release was observed. Increases to both the T(m) and DNase-I inhibition activity suggested interactions between E99K actin molecules under monomer-promoting conditions. Y166C actin had a higher critical concentration resulting in a lower Pi release rate due to reduced filament-forming potential. The locations of mutations on the ACTC protein correlated with the molecular effects; in general, mutations in subdomain 3 affected the stability of the ACTC protein or affect the polymerization of actin filaments, while mutations in subdomains 1 and 4 more likely affect protein-protein interactions.

Arginylation regulates intracellular actin polymer level by modulating actin properties and binding of capping and severing proteins.

Actin arginylation regulates lamella formation in motile fibroblasts, but the underlying molecular mechanisms are unknown. To understand how arginylation affects the actin cytoskeleton, we investigated the biochemical properties and the structural organization of actin filaments in wild-type and arginyltransferase (Ate1) knockout cells. We found that Ate1 knockout results in a dramatic reduction of the actin polymer levels in vivo accompanied by a corresponding increase in the monomer level. Purified nonarginylated actin has altered polymerization properties, and actin filaments from Ate1 knockout cells show altered interactions with several associated proteins. Ate1 knockout cells have severe impairment of cytoskeletal organization throughout the cell. Thus, arginylation regulates the ability of actin to form filaments in the whole cell rather than preventing the collapse of preformed actin networks at the cell leading edge as proposed in our previous model. This regulation is achieved through interconnected mechanisms that involve actin polymerization per se and through binding of actin-associated proteins.