Full-length Beta-subunit Research Articles

Ligand Selectivity of Soluble Guanylyl Cyclase: EFFECT OF THE HYDROGEN-BONDING TYROSINE IN THE DISTAL HEME POCKET ON BINDING OF OXYGEN, NITRIC OXIDE, AND CARBON MONOXIDE

Although soluble guanylyl cyclase (sGC) functions in an environment in which O(2), NO, and CO are potential ligands for its heme moiety, the enzyme displays a high affinity for only its physiological ligand, NO, but has a limited affinity for CO and no affinity for O(2). Recent studies of a truncated version of the sGC beta(1)-subunit containing the heme-binding domain (Boon, E. M., Huang, S H., and Marletta, M. A. (2005) Nat. Chem. Biol., 1, 53-59) showed that introduction of the hydrogen-bonding tyrosine into the distal heme pocket changes the ligand specificity of the heme moiety and results in an oxygen-binding sGC. The hypothesis that the absence of hydrogen-bonding residues in the distal heme pocket is sufficient to provide oxygen discrimination by sGC was put forward. We tested this hypothesis in a context of a complete sGC heterodimer containing both the intact alpha(1)- and beta(1)-subunits. We found that the I145Y substitution in the full-length beta-subunit of the sGC heterodimer did not produce an oxygen-binding enzyme. However, this substitution impeded the association of NO and destabilized the NO.heme complex. The tyrosine in the distal heme pocket also impeded both the binding and dissociation of the CO ligand. We propose that the mechanism of oxygen exclusion by sGC not only involves the lack of hydrogen bonding in the distal heme pocket, but also depends on structural elements from other domains of sGC.

Functional analysis of CK2beta-derived synthetic fragments.

Synthetic peptides reproducing the amino and carboxyl terminal region of CK2beta subunit have been analyzed for their ability to mimic different properties of full length beta subunit. Peptide beta[1-77], containing both the autophosphorylation site and the down-regulatory domain 55-64, is readily phosphorylated by alpha subunit whose activity is concomitantly inhibited. Such inhibition is accompanied by a weak interaction detectable by BIAcore sensograms but not by far Western blots, and is not reversed by polylysine which conversely overcome inhibition of calmodulin phosphorylation by full length beta subunit. A strong interaction with alpha is observed with beta[155-215] but not with its shorter derivative beta[170-215] as judged from far Western blotting and sucrose gradient ultracentrifugation analysis. Both peptides, however, affect the regular interaction between alpha and beta subunits altering the autophosphorylation pattern and responsiveness to salt. beta[155-215], unlike beta[170-215] tends to aggregate more readily than full length beta subunit. This behaviour which is reminiscent of the homodimerization of full length beta subunit, would indicate that tight self-association of beta[155-215] crucially depends on residues in the 155-170 sequence. Failure of beta[1-77] fragment to mediate responsiveness to polybasic peptides and accentuated self-association propensity of beta[155-215] suggest that other structural elements between the sequences 1-77 and 155-215 are required in order to confer optimal functionality to the beta subunit.

26 amino acids of an extracellular domain of the Na,K-ATPase alpha-subunit are sufficient for assembly with the Na,K-ATPase beta-subunit.

Chimeric cDNAs encoding a sarcoplasmic/endoplasmic reticulum Ca-ATPase (SERCA1) and regions of the Na,K-ATPase alpha-subunit were constructed to seek the minimal region of the alpha-subunit sufficient for assembly with the Na,K-ATPase beta-subunit. cDNAs encoding a chimera and the chicken beta-subunit were coexpressed in mammalian cells and assembly was assayed by immune precipitation of the chimeric subunit with a monoclonal antibody to the chicken beta-subunit. A chimera containing 26 amino acyl residues of the Na,K-ATPase alpha 1-subunit (NDVEDSYGQQWTFEQRKIVEFTCHTA) (Asn894 to Ala919) that replaced the corresponding avian SERCA1 Ca-ATPase amino acyl residues (Thr871 to Thr898) was able to assemble with the chicken beta-subunit. This alpha-subunit region is predicted to be extracellular, located between membrane-spanning domains 7 and 8 (H7-H8). Chimeras that assembled with full-length beta-subunit also assembled with a beta-subunit chimera that retained only the ectodomain of the chicken beta 1-subunit. These results suggest that the Na,K-ATPase alpha-subunit has the same topology in the membrane as the sarcoplasmic reticulum Ca-ATPase, probably with 10 membrane-spanning domains, and that the aminoacyl residues between membrane domains H7 and H8 are involved in assembly with the beta-subunit in the extracellular/lumenal space.

Mutational analysis of the consensus nucleotide binding sequences in the rat liver mitochondrial ATP synthase beta-subunit.

The coupling step in the biosynthesis of ATP in biological systems is generally believed to involve an energy-requiring release of ATP bound to the beta-subunit of the ATP synthase complex. A molecular description of the ATP binding site on the beta-subunit is, therefore, critical to understanding the mechanism of coupling in the enzyme. Previously, we reported that a purified, bacterially expressed rat liver beta-subunit binds adenine nucleotides tightly and specifically (Garboczi, D. N., Hullihen, J. H., and Pedersen, P. L. (1988) J. Biol. Chem. 263, 15694-15698). In order to assess the contribution of various regions of the isolated beta-subunit to the ATP binding site we have systematically deleted four different regions: the N-terminal region, the Walker A consensus region, the Walker B consensus region (Walker, J. E., Saraste, M., Runswick, M. J., and Gay, N. (1982) EMBO J. 1, 945-951), and a "C" region, which, like the A and B regions, bears homology to adenylate kinase. Plasmids directing the expression of double deletions of A and B regions, and B and C regions were also constructed. In addition, 2 residues outside of these regions, His-177 and Tyr-345, which have been predicted to play a central role in nucleotide binding, were mutated. Rabbit antisera to synthetic peptides of the A and C regions verified the identity of the bacterially expressed mutant proteins. Seven of the eight mutant proteins overexpressed in Escherichia coli were resistant to E. coli proteases in the preparative stages, as predicted for compact folded proteins. Furthermore, circular dichroism spectropolarimetry revealed no profound structural alterations in the purified mutant proteins. Relative to the overexpressed full-length beta-subunit, the mutant lacking the A consensus region suffered a 30-fold loss of affinity for ATP and a loss of specificity for 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate (TNP-ATP) over 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-monophosphate. The mutant proteins lacking either the N-terminal region or the B region exhibited nucleotide binding properties similar to the full-length beta-subunit, whereas the mutant protein lacking the C region suffered an order of magnitude reduction in affinity for ATP. The affinity of the A and B region double deletion was indistinguishable from the A region deletion in regard to TNP-ATP binding, while the double deletion mutant lacking the B and C regions was not stably expressed in the E. coli SE6004.(ABSTRACT TRUNCATED AT 400 WORDS)

Cloning of the H,K-ATPase beta subunit. Tissue-specific expression, chromosomal assignment, and relationship to Na,K-ATPase beta subunits.

We have isolated cDNA clones encoding the bovine and rat gastric H,K-ATPase beta subunit. A bovine abomasum lambda gt11 cDNA library was screened with a monoclonal antibody raised against the rabbit H,K-ATPase beta subunit. A single positive phage clone containing an approximately 900-base pair cDNA insert was identified as reactive with the antibody. The identity of the cDNA was established by comparing the deduced amino acid sequence with sequences of cyanogen bromide fragments of the porcine H,K-ATPase beta subunit. Polymerase chain reaction and rapid amplification of cDNA ends were used to generate a cDNA fragment encoding the carboxyl-terminal portion of the rat gastric H,K-ATPase beta subunit. A rat stomach cDNA library was screened with the polymerase chain reaction product, and several full-length beta subunit cDNA clones were identified. The open reading frame predicts a protein of 294 amino acids with a molecular weight of 33,689. The rat H,K-ATPase beta subunit shows 41% amino acid sequence identity to the rat Na,K-ATPase beta 2 subunit and shares a number of structural similarities with Na,K-ATPase beta subunit isoforms. By analyzing the segregation of restriction fragment length polymorphisms among recombinant inbred strains of mice, we localized the H,K-ATPase beta subunit gene to murine chromosome 8. Northern and Western blot analysis reveals that this gene is expressed exclusively in stomach. Our results suggest that the H,K-ATPase and Na,K-ATPase beta subunits evolved from a common ancestral gene and may play similar functional roles in enzyme activity.