Experimental dissection and characterization of the functional domains in cardiac troponin C

Contraction of vertebrate cardiac muscle is regulated by the binding of Ca$\sp{2+}$ to the troponin C (cTnC) subunit of the troponin complex. In this study, we have used site-directed mutagenesis and a variety of assay techniques to explore the functional roles of regions in cTnC, including Ca$\sp{2+}$/Mg$\sp{2+}$-binding sites III and IV, the functionally inactive site I, the N-terminal helix, the N-terminal hydrophobic pocket and the two cysteine residues with regard to their ability to form disulfide bonds. Conversion of the first Ca$\sp{2+}$ ligand from Asp to Ala inactivated sites III and IV and decreased the apparent affinity of cTnC for the thin filament. Conversion of the second ligand from Asn to Ala also inactivated these sites in the free protein but Ca$\sp{2+}$-binding was recovered upon association with troponin I and troponin T. The Ca$\sp{2+}$-concentrations required for tight thin filament-binding by proteins containing second-ligand mutations were significantly greater than that required for the wild-type protein. Mutation of site I such that the primary sequence was that of an active site with the first Ca$\sp{2+}$ ligand changed from Asp to Ala resulted in a 70% decrease in maximal Ca$\sp{2\sp+}$ dependent ATPase activity in both cardiac and fast skeletal myofibrils. Thus, the primary sequence of the inactive site I in cTnC is functionally important. Major changes in the sequence of the N-terminus had little effect on the ability of cTnC to recover maximal activity but deletion of the first nine residues resulted in a 60 to 80% decrease in maximal activity with only a minor decrease in the pCa$\sb{50}$ of activation, suggesting that the N-terminal helix must be present but that a specific sequence is not required. The formation of an inter- or intramolecular disulfide bonds caused the exposure of hydrophobic surfaces on cTnC and rendered the protein Ca$\sp{2+}$ independent. Finally, elution patterns from a hydrophobic interactions column suggest that cTnC undergoes a significant change in hydrophobicity upon Ca$\sp{2+}$ binding, the majority of which is caused by site II. These latter data show an interesting correlation between exposure of hydrophobic surfaces on and activation of cTnC. Overall, these results represent significant progress toward the elucidation of the functional roles of a variety of structural regions in cTnC. ^

Functions and intramolecular interactions of ribosomal RNA in decoding of termination codons

Two regions in the 3$\prime$ domain of 16S rRNA (the RNA of the small ribosomal subunit) have been implicated in decoding of termination codons. Using segment-directed PCR random mutagenesis, I isolated 33 translational suppressor mutations in the 3$\prime$ domain of 16S rRNA. Characterization of the mutations by both genetic and biochemical methods indicated that some of the mutations are defective in UGA-specific peptide chain termination and that others may be defective in peptide chain termination at all termination codons. The studies of the mutations at an internal loop in the non-conserved region of helix 44 also indicated that this structure, in a non-conserved region of 16S rRNA, is involved in both peptide chain termination and assembly of 16S rRNA.^ With a suppressible trpA UAG nonsense mutation, a spontaneously arising translational suppressor mutation was isolated in the rrnB operon cloned into a pBR322-derived plasmid. The mutation caused suppression of UAG at two codon positions in trpA but did not suppress UAA or UGA mutations at the same trpA positions. The specificity of the rRNA suppressor mutation suggests that it may cause a defect in UAG-specific peptide chain termination. The mutation is a single nucleotide deletion (G2484$\Delta$) in helix 89 of 23S rRNA (the large RNA of the large ribosomal subunit). The result indicates a functional interaction between two regions of 23S rRNA. Furthermore, it provides suggestive in vivo evidence for the involvement of the peptidyl-transferase center of 23S rRNA in peptide chain termination. The $\Delta$2484 and A1093/$\Delta$2484 (double) mutations were also observed to alter the decoding specificity of the suppressor tRNA lysT(U70), which has a mutation in its acceptor stem. That result suggests that there is an interaction between the stem-loop region of helix 89 of 23S rRNA and the acceptor stem of tRNA during decoding and that the interaction is important for the decoding specificity of tRNA.^ Using gene manipulation procedures, I have constructed a new expression vector to express and purify the cellular protein factors required for a recently developed, realistic in vitro termination assay. The gene for each protein was cloned into the newly constructed vector in such a way that expression yielded a protein with an N-terminal affinity tag, for specific, rapid purification. The amino terminus was engineered so that, after purification, the unwanted N-terminal tag can be completely removed from the protein by thrombin cleavage, yielding a natural amino acid sequence for each protein. I have cloned the genes for EF-G and all three release factors into this new expression vector and the genes for all the other protein factors into a pCAL-n expression vector. These constructs will allow our laboratory group to quickly and inexpensively purify all the protein factors needed for the new in vitro termination assay. (Abstract shortened by UMI.) ^

Regulation of Alix by an autoinhibitory intramolecular interaction

In eukaryotic cells, the ESCRTs (endosomal sorting complexes required for transport) machinery is required for cellular processes such as endosomal sorting, retroviral budding and cytokinesis. The ALG-2 interacting protein Alix is a modular adaptor protein that is critically involved in these ESCRTs-associated cellular processes and consists of an N-terminal Bro1 domain, a middle V domain and C-terminal Pro-rich domain (PRD). In these cellular processes, Alix interacts with the ESCRT-III component CHMP4 at the Bro1 domain, with HIV-1 p6 Gag or EIAV p9Gag at the V domain, and with the ESCRT-I component TSG101 at the Pro-rich domain. Here we demonstrate that the N-terminal Bro1 domain forms an intramolecular interaction with C-terminal PRD within Alix. This Bro1-PRD intramolecular interaction forms a closed conformation of Alix that autoinhibits Alix interaction with all of these partner proteins. Moreover, the binding of Ca2+-activated ALG-2 to the PRD of Alix relieves the autoinhibitory intramolecular interaction, resulting in an open conformation of Alix which is able to interact with all of these partner proteins. The partner proteins bound to Alix in turn maintain Alix in the open conformation after ALG-2 dissociation with Alix. Consistent with the effect of Ca2+-activated ALG-2 on opening/activating Alix in these ESCRTs-associated functions, ALG-2 overexpression accelerates EGF-induced degradation of EGFR in an Alix-dependent manner. These findings discover an intrinsic autoinhibitory mechanism of Alix and a two-step process to activate/open Alix and then keep Alix active/open. This study has solved long-standing issues on the regulations of Alix in ESCRTs-associated functions and the role of ALG-2-Alix interaction, and may serve as the structural basis for further studies about Alix regulations. ^