The Catalytic Transition State in ATP Synthase

The catalytic transition state of ATP synthase has been characterized and modeled by combined use of (1) Mg-ADP–fluoroaluminate, Mg-ADP–fluoroscandium, and corresponding Mg-IDP–fluorometals as transition-state analogs; (2) fluorescence signals of -Trp331 and -Trp148 as optical probes to assess formation of the transition state; (3) mutations of critical catalytic residues to determine side-chain ligands required to stabilize the transition state. Rate acceleration by positive catalytic site cooperativity is explained as due to mobility of -Arg376, acting as an arginine finger residue, which interacts with nucleotide specifically at the transition state step of catalysis, not with Mg-ATP- or Mg-ADP-bound ground states. We speculate that formation and collapse of the transition state may engender catalytic site / subunit-interface conformational movement, which is linked to -subunit rotation.     

Parasites and stable isotopes: a comparative analysis of isotopic discrimination in parasitic trophic interactions

Stable isotopes are widely used to identify trophic interactions and to determine trophic positions of organisms in food webs. Comparative studies have provided general insights into the variation in isotopic composition between consumers and their diet (discrimination factors) in predator–prey and herbivore–plant relationships while other major components of food webs such as host–parasite interactions have been largely overlooked. In this study, we conducted a literature‐based comparative analysis using phylogenetically‐controlled mixed effects models, accounting for both parasite and host phylogenies, to investigate patterns and potential drivers in Δ¹³C and Δ¹⁵N discrimination factors in metazoan parasitic trophic interactions. Our analysis of 101 parasite–host pairs revealed a large range in Δ¹³C (–8.2 to 6.5) and Δ¹⁵N (–6.7 to 9.0) among parasite species, with no significant overall depletion or enrichment of ¹³C and ¹⁵N in parasites. As previously found in other trophic interactions, we identified a scaling relationship between the host isotopic value and both discrimination factors with Δ¹³C and Δ¹⁵N decreasing with increasing host δ¹³C and δ¹⁵N, respectively. Furthermore, parasite phylogenetic history explained a large fraction (>60%) of the observed variation in the Δ¹⁵N discrimination factor. Our findings suggest that the traditional isotope ecology framework (using an average Δ¹⁵N of 3.4‰) applies poorly to parasitic trophic interactions. They further indicate the need for a scaled rather than a fixed trophic discrimination factor framework along gradients of host δ¹⁵N. We also identified several conceptual and methodological issues which should to be considered in future research to help integrate parasitic interactions into a holistic isotope ecology framework across diverse trophic interactions.     

Mutational analysis of conserved aromatic residues in the A-loop of the ABC transporter ABCB1A (mouse Mdr3)

The A-loop is a recently described conserved region in the NBDs of ABC transporters [Ambudkar, S.V., Kim, I.-W., Xia, D. and Sauna, Z.E. (2006) The A-loop, a novel conserved aromatic acid subdomain upstream of the Walker A motif in ABC transporters, is critical for ATP binding. FEBS Lett. 580, 1049-1055; Kim, I.W., Peng, X.H., Sauna, Z.E., FitzGerald, P.C., Xia, D., Muller, M., Nandigama, K. and Ambudkar, S.V. (2006) The conserved tyrosine residues 401 and 1044 in ATP sites of human P-glycoprotein are critical for ATP binding and hydrolysis: evidence for a conserved subdomain, the A-loop in the ATP-binding cassette. Biochemistry 45, 7605-7616]. In mouse P-glycoprotein (Abcb1a), the aromatic residue of the A-loop in both NBDs is a tyrosine: Y397 in NBD1 and Y1040 in NBD2. Another tyrosine residue (618 in NBD1 and 1263 in NBD2) also appears to lie in proximity to the ATP molecule. We have mutated residues Y397, Y618, Y1040, and Y1263 to tryptophan and analyzed the effect of these substitutions on transport properties, ATP binding, and ATP hydrolysis by Abcb1a (mouse Mdr3). Y618W and Y1263W enzymes had catalytic characteristics similar to WT Abcb1a. On the other hand, Y397W and Y1040W showed impaired transport and greatly reduced ATPase activity, including a approximately 10-fold increase in Km for MgATP. Thus, Y397 and Y1040 play an important role in Abcb1a catalysis.     

Membrane type 1 matrix metalloproteinase is necessary for distal airway epithelial repair and keratinocyte growth factor receptor expression after acute injury

Membrane type 1 matrix metalloproteinase (MT1-MMP) is a protease produced by airway epithelial cells in various diseases. Since other MMPs are involved in bronchial epithelial repair, we investigated the role of MT1-MMP in naphthalene-induced small airway injury and repair in wild-type (WT) and MT1-MMP-knockout (KO) mice. The degree of injury was similar in both strains, but the MT1-MMP KO mice were unable to reconstitute a normal, fully differentiated airway epithelium 28 days after injury. MT1-MMP was required for the proliferative response in distal airway epithelial cells, resulting in decreased cell density and airway epithelial cell differentiation in MT1-MMP KO mice. Surprisingly, EGF-mediated signaling was unaltered in MT1-MMP KO mice and therefore unrelated to the proliferative response. However, keratinocyte growth factor receptor (KGFR) expression was significantly upregulated before the proliferative response and markedly less evident in the distal airway epithelium of MT1-MMP KO mice. These results indicate MT1-MMP is involved in KGFR expression and epithelial cell proliferation after acute airway injury.     

The influences of hinge length and composition on the susceptibility of human IgA to cleavage by diverse bacterial IgA1 proteases

The influences of IgA hinge length and composition on its susceptibility to cleavage by bacterial IgA1 proteases were examined using a panel of IgA hinge mutants. The IgA1 proteases of Streptococcus pneumoniae, Streptococcus sanguis strains SK4 and SK49, Neisseria meningitidis, Neisseria gonorrhoeae, and Haemophilus influenzae cleaved IgA2-IgA1 half hinge, an Ab featuring half of the IgA1 hinge incorporated into the equivalent site in IgA1 protease-resistant IgA2, whereas those of Streptococcus mitis, Streptococcus oralis, and S. sanguis strain SK1 did not. Hinge length reduction by removal of two of the four C-terminal proline residues rendered IgA2-IgA1 half hinge resistant to all streptococcal IgA1 metalloproteinases but it remained sensitive to cleavage by the serine-type IgA1 proteases of Neisseria and Haemophilus spp. The four C-terminal proline residues could be substituted by alanine residues or transferred to the N-terminal extremity of the hinge without affect on the susceptibility of the Ab to cleavage by serine-type IgA1 proteases. However, their removal rendered the Ab resistant to cleavage by all the IgA1 proteases. We conclude that the serine-type IgA1 proteases of Neisseria and Haemophilus require the Fab and Fc regions to be separated by at least ten (or in the case of N. gonorrhoeae type I protease, nine) amino acids between Val(222) and Cys(241) (IgA1 numbering) for efficient access and cleavage. By contrast, the streptococcal IgA1 metalloproteinases require 12 or more appropriate amino acids between the Fab and Fc to maintain a minimum critical distance between the scissile bond and the start of the Fc.     

Characterization of Escherichia coli ATP synthase beta-subunit mutations using a chromosomal deletion strain.

(1) We constructed Escherichia coli strain JP17 with a deletion in the ATP synthase beta-subunit gene. JP17 is completely deficient in ATP synthase activity and expresses no beta-subunit. Expression of normal beta-subunit from a plasmid restores haploid levels of ATP synthase in membranes. JP17 was shown to be efficacious for studies of beta-subunit mutations. Site-directed mutants were studied directly in JP17. Randomly generated chromosomal mutants were identified by PCR and DNA sequencing, cloned, and expressed in JP17. (2) Eight novel mutations occurring within the putative catalytic nucleotide-binding domain were characterized with respect to their effects on catalysis and structure. The mutations beta C137S, beta G152D, beta G152R, beta E161Q, beta E161R, and beta G251D each impaired catalysis without affecting enzyme assembly or oligomeric structure and are of interest for future studies of catalytic mechanism. The mutations beta D301V and beta D302V, involving strongly conserved carboxyl residues, caused oligomeric instability of F1. However, growth characteristics of these mutants suggested that neither carboxyl side chain is critical for catalysis. (3) The mutations beta R398C and beta R398W rendered ATP synthase resistant to aurovertin, giving strong support to the view that beta R398 is a key residue in the aurovertin-binding site. Neither beta R398C or beta R398W impaired catalysis significantly.     

The effect of temperature on the synthesis and assembly of proticine 3 particles by Proteus mirabilis

Proteus mirabilis CW977 produced high yields of the bacteriocin proticine 3 upon mitomycin C induction of cultures growing at 30 degrees C. The proticine was purified and found to have a relative density of 1.299 and to be composed of 10 proteins assembled into structures resembling contractile phage tails. When induction was performed at 41 degrees C neither proticine particles nor proticine activity was detected, although the growth rate of cells and degree of lysis were indistinguishable from that at 30 degrees C. Failure in proticine production was due to a 41 degrees C sensitive stage occurring between 60 and 90 min after the addition of mitomycin C. During this period at 30 degrees C, two proteins of mol. wt 58 000 and 41 000 were formed. These proteins were associated with events leading to the formation of proticine particles with biological activity. When the production of both proteins was prevented either by chloramphenicol or as a result of mutation or through sampling before they were formed, no proticine particles were found nor proticine activity detected. The synthesis of both proteins was also inhibited at 41 degrees C. Co-electrophoresis of the labelled proteins with unlabelled purified proticine confirmed that the protein of mol. wt 58 000 was a proticine structural protein. The protein of mol. wt 41 000 was not a structural component of proticine and its role, if any, in proticine 3 production is possibly that of an assembly protein.