Patrick R. Connelly

Nucleotide-binding Domains of Cystic Fibrosis Transmembrane Conductance Regulator, an ABC Transporter, Catalyze Adenylate Kinase Activity but Not ATP Hydrolysis

The cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel in the ATP-binding cassette (ABC) transporter family. CFTR consists of two transmembrane domains, two nucleotide-binding domains (NBD1 and NBD2), and a regulatory domain. Previous biochemical reports suggest NBD1 is a site of stable nucleotide interaction with low ATPase activity, whereas NBD2 is the site of active ATP hydrolysis. It has also been reported that NBD2 additionally possessed adenylate kinase (AK) activity. Knowledge about the intrinsic biochemical activities of the NBDs is essential to understanding the Cl– ion gating mechanism. We find that purified mouse NBD1, human NBD1, and human NBD2 function as adenylate kinases but not as ATPases. AK activity is strictly dependent on the addition of the adenosine monophosphate (AMP) substrate. No liberation of [33P]phosphate is observed from the γ-33P-labeled ATP substrate in the presence or absence of AMP. AK activity is intrinsic to both human NBDs, as the Walker A box lysine mutations abolish this activity. At low protein concentration, the NBDs display an initial slower nonlinear phase in AK activity, suggesting that the activity results from homodimerization. Interestingly, the G551D gating mutation has an exaggerated nonlinear phase compared with the wild type and may indicate this mutation affects the ability of NBD1 to dimerize. hNBD1 and hNBD2 mixing experiments resulted in an 8–57-fold synergistic enhancement in AK activity suggesting heterodimer formation, which supports a common theme in ABC transporter models. A CFTR gating mechanism model based on adenylate kinase activity is proposed.

BINDING OF OXYGEN AND CARBON-MONOXIDE TO THE HEMOCYANIN FROM THE SPINY LOBSTER

A high precision, two-dimensional study of oxygen and carbon monoxide binding to Panulirus interruptus hemocyanin has been carried out. Global data analysis of three types of experiments, probing the molecule in its various states of CO and O2 ligation, revealed the entire hexamer to be the basic allosteric unit involved in a two-state mechanism. The co-operativity and linkage of the two ligands are presented in terms of derivative Hill plot surfaces extended along co-ordinates of CO and O2 activities giving a detailed and comprehensive view of the binding behavior. Among the findings is an apparent high co-operativity of carbon monoxide binding at high oxygen activity. The results are discussed in view of a general mechanism for co-operative behavior found in larger hemocyanin aggregates concerning nested allosteric interactions.

Analysis and parameter resolution in highly cooperative systems

We have examined common methods of analysis of highly cooperative systems such as oxygen binding by hemoglobin and thermal denaturation. Through extensive simulation of ligand-binding data for a tetrameric macromolecule we show that careful attention must be paid to the formulation of the fitting function and to proper assessment of the number of parameters involved. We conclude that the partition function should be formulated in terms of overall reaction parameters as opposed to stepwise reaction parameters and that bias is introduced by fixing physical parameters such as extrapolated end points.

Analysis of zeros of binding polynomials for tetrameric hemoglobins

A quantitative measure of the validity of the MWC description of cooperative binding equilibria has been obtained which uses only the Adair constants. This is accomplished through simple relationships using the zeros of the Adair binding polynomial and unique properties of the zeros of MWC polynomials as described in the accompanying paper (W.E. Briggs, Biophys. Chem. 24 (1986) 311). The method is applied to oxygen binding to a large number of hemoglobins under a wide variety of conditions. In most cases, exemplified by human hemoglobin under a wide range of conditions, the MWC model is allowed and the probability of its suitability is determined. The probability given by this method correlates directly with the deviation between the experimental binding curve and that derived from the theory. In several cases the pattern of the Adair polynomial zeros immediately excludes the MWC model, most notably for carp hemoglobins. A physical picture of cooperative binding site interactions is nevertheless obtained from the patterns of zeros as they relate to the factorization of the binding polynomial.