Molecular basis of the cooperative binding of Cu(I) and Cu(II) to the CopK protein from Cupriavidus metallidurans CH34

The bacterium Cupriavidus metallidurans CH34 is resistant to high environmental concentrations of many metal ions. Upon copper challenge, it upregulates the periplasmic protein CopK (8.3 kDa). The function of CopK in the copper resistance response is ill-defined, but CopK demonstrates an intriguing cooperativity: occupation of a high-affinity Cu(I) binding site generates a high-affinity Cu(II) binding site, and the high-affinity Cu(II) binding enhances Cu(I) binding. Native CopK and targeted variants were examined by chromatographic, spectroscopic, and X-ray crystallographic probes. Structures of two distinct forms of Cu(I)Cu(II)-CopK were defined, and structural changes associated with occupation of the Cu(II) site were demonstrated. In solution, monomeric Cu(I)Cu(II)-CopK features the previously elucidated Cu(I) site in Cu(I)-CopK, formed from four S(δ) atoms of Met28, -38, -44, and -54 (site 4S). Binding of Cu(I) to apo-CopK induces a conformational change that releases the C-terminal β-strand from the β-sandwich structure. In turn, this allows His70 and N-terminal residues to form a large loop that includes the Cu(II) binding site. In crystals, a polymeric form of Cu(I)Cu(II)-CopK displays a Cu(I) site defined by the S(δ) atoms of Met26, -38, and -54 (site 3S) and an exogenous ligand (modeled as H(2)O) and a Cu(II) site that bridges dimeric CopK molecules. The 3S Cu(I) binding mode observed in crystals was demonstrated in solution in protein variant M44L where site 4S is disabled. The intriguing copper binding chemistry of CopK provides molecular insight into Cu(I) transfer processes. The adaptable nature of the Cu(I) coordination sphere in methionine-rich clusters allows copper to be relayed between clusters during transport across membranes in molecular pumps such as CusA and Ctr1.     

Exclusion of the native alpha-helix from the amyloid fibrils of a mixed alpha/beta protein

Members of the cystatin superfamily are involved in an inherited form of cerebral amyloid angiopathy and readily form amyloid fibrils in vitro. We have determined the structured core of human stefin B (cystatin B) amyloid fibrils using quenched hydrogen exchange and NMR. The core contains residues from four of the five strands of the native β-sheet, delimited by unprotected loop regions analogous to those of the native monomeric structure. However, non-native features are also apparent, the most striking of which is the exclusion of the native α-helix. Before forming amyloid in vitro, cystatins dimerise via 3D domain swapping, and assemble into tetramers with trans to cis isomerism of a conserved proline. In the fibril, the hinge loop that forms an extended β-structure in the dimer remains protected, consistent with the domain-swapping interface being maintained. However, the fibril data are not compatible with a simple 3D domain-swapping model for amyloid formation, and the displacement of the helix points to alternative packing arrangements of native-like β-structure, in which proline isomerism is important in preventing steric clashing.     

Structure of the leech protein saratin and characterization of its binding to collagen

The leech protein Saratin from Hirudo medicinalis prevents thrombocyte aggregation by interfering with the first binding step of the thrombocytes to collagen by binding to collagen. We solved the three-dimensional structure of the leech protein Saratin in solution and identified its collagen binding site by NMR titration experiments. The NMR structure of Saratin consists of one α-helix and a five-stranded β-sheet arranged in the topology ββαβββ. The C-terminal region, of about 20 amino acids in length, adopts no regular structure. NMR titration experiments with collagen peptides show that the collagen interaction of Saratin takes place in a kind of notch that is formed by the end of the α-helix and the β-sheet. NMR data-driven docking experiments to collagen model peptides were used to elucidate the putative binding mode of Saratin and collagen. Mainly, parts of the first and the end of the fifth β-strand, the loop connecting the α-helix and the third β-strand, and a short part of the loop connecting the fourth and fifth β-strand participate in binding.     

Crowding agents and osmolytes provide insight into the formation and dissociation of RNase A oligomers

RNase A forms 3D domain-swapped oligomers with novel enzymatic and biological activities. We study how crowding agents and osmolytes affect the formation and dissociation of RNase A oligomers. The crowding agents Ficoll and dextran were found to enhance oligomer formation, whereas the stabilizers sodium sulfate, glycine and trimethylammonium oxide (TMAO) do not. In contrast, TMAO significantly slows RNase A dimer dissociation, while the effect of Ficoll is small. These results lead us to propose that the mechanisms of oligomer formation and dissociation are different. In the RNase A “C-dimer”, the C-terminal β-strand is swapped between two subunits. The loop preceding this β-strand adopts a β-sheet which has been proposed to resemble amyloid structurally. Hydrogen/deuterium (H/D) exchange of the RNase A C-dimer reveal that the H-bonds formed between the swapped C-terminal β-strand and the other subunit are strong. Their rupture may be crucial for C-dimer dissociation. In contrast, H-bonds formed by Asn 113 in the novel β-sheet adopted by the hinge loop in the C-dimer are not strongly protected. Besides the fundamental insights obtained, the results represent a technical advance for obtaining increased oligomer yields and storage lifetimes.     

An Integrative Approach Combining Noncovalent Mass Spectrometry, Enzyme Kinetics and X-ray Crystallography to Decipher Tgt Protein-Protein and Protein-RNA Interaction

The tRNA-modifying enzyme tRNA-guanine transglycosylase (Tgt) is a putative target for new selective antibiotics against Shigella bacteria. The formation of a Tgt homodimer was suggested on the basis of several crystal structures of Tgt in complex with RNA. In the present study, noncovalent mass spectrometry was used (i) to confirm the dimeric oligomerization state of Tgt in solution and (ii) to evidence the binding stoichiometry of the complex formed between Tgt and its full-length substrate tRNA. To further investigate the importance of Tgt protein-protein interaction, point mutations were introduced into the dimer interface in order to study their influence on the formation of the catalytically active complex. Enzyme kinetics revealed a reduced catalytic activity of these mutated variants, which could be related to a destabilization of the dimer formation as evidenced by both noncovalent mass spectrometry and X-ray crystallography. Finally, the effect of inhibitor binding was investigated by noncovalent mass spectrometry, thus providing the binding stoichiometries of Tgt:inhibitor complexes and showing competitive interactions in the presence of tRNA. Inhibitors that display an influence on the formation of the dimer interface in the crystal structure are promising candidates to alter the protein-protein interaction, which could provide a new way to inhibit Tgt.     

Positively Cooperative Binding of Zinc Ions to Bacillus cereus 569/H/9 β-Lactamase II Suggests that the Binuclear Enzyme Is the Only Relevant Form for Catalysis

Metallo-β-lactamases catalyze the hydrolysis of most β-lactam antibiotics and hence represent a major clinical concern. While enzymes belonging to subclass B1 have been shown to display maximum activity as dizinc species, the actual metal-to-protein stoichiometry and the affinity for zinc are not clear. We have further investigated the process of metal binding to the β-lactamase II from Bacillus cereus 569/H/9 (known as BcII). Zinc binding was monitored using complementary biophysical techniques, including circular dichroism in the far-UV, enzymatic activity measurements, competition with a chromophoric chelator, mass spectrometry, and nuclear magnetic resonance. Most noticeably, mass spectrometry and nuclear magnetic resonance experiments, together with catalytic activity measurements, demonstrate that two zinc ions bind cooperatively to the enzyme active site (with K₁/K₂ ≥ 5) and, hence, that catalysis is associated with the dizinc enzyme species only. Furthermore, competitive experiments with the chromophoric chelator Mag-Fura-2 indicates K₂ < 80 nM. This contrasts with cadmium binding, which is clearly a noncooperative process with the mono form being the only species significantly populated in the presence of 1 molar equivalent of Cd(II). Interestingly, optical measurements reveal that although the apo and dizinc species exhibit undistinguishable tertiary structural organizations, the metal-depleted enzyme shows a significant decrease in its α-helical content, presumably associated with enhanced flexibility.     

The Metalloregulatory Zinc Site in Streptococcus pneumoniae AdcR, a Zinc-activated MarR Family Repressor

Streptococcus pneumoniae D39 AdcR (adhesin competence repressor) is the first metal-sensing member of the MarR (multiple antibiotic resistance repressor) family to be characterized. Expression profiling with a ΔadcR strain grown in liquid culture (brain-heart infusion) under microaerobic conditions revealed upregulation of 13 genes, including adcR and adcCBA, encoding a high-affinity ABC uptake system for zinc, and genes encoding cell-surface zinc-binding pneumococcal histidine triad (Pht) proteins and AdcAII (Lmb, laminin binding). The ΔadcR, H108Q and H112Q adcR mutant allelic strains grown in 0.2 mM Zn(II) exhibit a slow-growth phenotype and an approximately twofold increase in cell-associated Zn(II). Apo- and Zn(II)-bound AdcR are homodimers in solution and binding to a 28-mer DNA containing an adc operator is strongly stimulated by Zn(II) with K_DNA-Zn = 2.4 × 10⁸ M^- 1 (pH 6.0, 0.2 M NaCl, 25 °C). AdcR binds two Zn(II) per dimer, with stepwise Zn(II) affinities K_Zn1 and K_Zn2 of ≥ 10⁹ M^- 1 at pH 6.0 and ≥ 10¹² M^- 1 at pH 8.0, and one to three lower affinity Zn(II) depending on the pH. X-ray absorption spectroscopy of the high-affinity site reveals a pentacoordinate N/O complex and no cysteine coordination, the latter finding corroborated by wild type-like functional properties of C30A AdcR. Alanine substitution of conserved residues His42 in the DNA-binding domain, and His108 and His112 in the C-terminal regulatory domain, abolish high-affinity Zn(II) binding and greatly reduce Zn(II)-activated binding to DNA. NMR studies reveal that these mutants adopt the same folded conformation as dimeric wild type apo-AdcR, but fail to conformationally switch upon Zn(II) binding. These studies implicate His42, His108 and H112 as metalloregulatory zinc ligands in S. pneumoniae AdcR.     

Reversible unfolding of dimeric phosphofructokinase-2 from Escherichia coli reveals a dominant role of inter-subunit contacts for stability

Escherichia coli phosphofructokinase-2 (Pfk-2) is a homodimer whose subunits consist of a large domain and an additional β-sheet that provides the interfacial contacts between the subunits, creating a β-barrel flattened-like structure with the adjacent subunit’s β-sheet. To determine how the structural organization of Pfk-2 determines its stability, the reversible unfolding of the enzyme was characterized under equilibrium conditions by enzymatic activity, circular dichroism, fluorescence and hydrodynamic measurements. Pfk-2 undergoes a cooperative unfolding/dissociation process with the accumulation of an expanded and unstructured monomeric intermediate with a marginal stability and a large solvent accessibility with respect to the native dimer.     

Solution Structure of Human ζ-COP: Direct Evidences for Structural Similarity between COP I and Clathrin-Adaptor Coats

COP-I-coated vesicles are protein and lipid carriers that mediate intra-Golgi transport and transport from the cis-Golgi complex to the endoplasmic reticulum in cells. The coatomer of the vesicles coat is comprised of seven subunits: α-COP, ?-COP, β′-COP, β-COP, γ-COP, δ-COP, and ζ-COP. Here we report the solution structure of a truncated form (residues 1-149; ζ-COP149) of human ζ-COP (total 177 residues). It is the first three-dimensional structure of a “core” subunit of the COP I F-subcomplex. The structure of ζ-COP149 mainly consists of a disordered N-terminal tail, a five-stranded antiparallel β-sheet, a two-stranded antiparallel β-sheet, and five α-helices. The global folding of ζ-COP149 is very similar to the crystal structures of AP1-σ1 and AP2-σ2, directly demonstrating the structural similarity between the “core” subunits of the COP I F-subcomplex and adaptor protein complexes. Through structural comparison and mutagenesis study, we have also demonstrated that the heterodimers of ζ-COP149 and γ-COP have packing interfaces and relative subunit orientations similar to those of AP2-σ2 and AP2-α heterodimers. These results provide direct evidence supporting the previous proposal that the COP I F-subcomplex and adaptor protein complexes have similar tertiary and quaternary structures.     

Interaction of Alzheimer's A beta peptide with an engineered binding protein--thermodynamics and kinetics of coupled folding-binding

The oligomerization and aggregation of the amyloid-β (Aβ) peptide, a cleavage product of the amyloid precursor protein predominantly 40 or 42 amino acids in length, has been implicated in the pathogenesis of Alzheimer's disease. The identification of Aβ-binding agents, e.g., antibodies or peptides, constitutes a promising therapeutic approach. However, the amount of structural and biophysical data on the underlying Aβ interactions is currently very limited. We have earlier determined the structure of Aβ(1-40) in complex with the affibody protein Z_Aβ3, a selected binding protein based on a three-helix bundle scaffold (Z domain). Z_Aβ3 is a dimer of affibody subunits linked via a disulfide bridge involving a selected cysteine mutation at position 28. Z_Aβ3 binds to the central and C-terminal part of Aβ (residues 17-36), which adopts a β-hairpin conformation in the complex. Here we present a detailed biophysical analysis of the Z_Aβ3:Aβ(1-40) interaction, employing NMR, circular dichroism spectroscopy, 8-anilino-1-naphthalenesulfonic acid and tyrosine fluorescence, size-exclusion chromatography, thermal denaturation profiles and isothermal titration calorimetry. We conclude that (i) free Z_Aβ3 is characterized by conformational exchange and the loss of helix 1 of the three-helix bundle scaffold; (ii) a high-energy barrier is associated with the conversion of an initial Z_Aβ3:Aβ(1-40) recognition complex into the native complex structure, entailing slow binding kinetics; (iii) both Aβ and Z_Aβ3 fold upon binding, which, e.g., becomes manifest in the binding thermodynamics that feature a large negative change in heat capacity; (iv) the C28-disulfide does not merely afford dimerization, but its impact on the binding interfaces of the affibody subunits and Aβ is a prerequisite for tight binding. The extensive folding coupled to binding observed here likely constitutes an obligate feature of biomolecular interactions involving the central and C-terminal part of Aβ. Options for improvement of Z_Aβ binding proteins are discussed.     

Structure, folding and stability of FimA, the main structural subunit of type 1 pili from uropathogenic Escherichia coli strains

Filamentous type 1 pili are responsible for attachment of uropathogenic Escherichia coli strains to host cells. They consist of a linear tip fibrillum and a helical rod formed by up to 3000 copies of the main structural pilus subunit FimA. The subunits in the pilus interact via donor strand complementation, where the incomplete, immunoglobulin-like fold of each subunit is complemented by an N-terminal donor strand of the subsequent subunit. Here, we show that folding of FimA occurs at an extremely slow rate (half-life: 1.6 h) and is catalyzed more than 400-fold by the pilus chaperone FimC. Moreover, FimA is capable of intramolecular self-complementation via its own donor strand, as evidenced by the loss of folding competence upon donor strand deletion. Folded FimA is an assembly-incompetent monomer of low thermodynamic stability (− 10.1 kJ mol^− 1) that can be rescued for pilus assembly at 37 °C because FimC selectively pulls the fraction of unfolded FimA molecules from the FimA folding equilibrium and allows FimA refolding on its surface. Elongation of FimA at the C-terminus by its own donor strand generated a self-complemented variant (FimAa) with alternative folding possibilities that spontaneously adopts the more stable conformation (− 85.0 kJ mol^− 1) in which the C-terminal donor strand is inserted in the opposite orientation relative to that in FimA. The solved NMR structure of FimAa revealed extensive β-sheet hydrogen bonding between the FimA pilin domain and the C-terminal donor strand and provides the basis for reconstruction of an atomic model of the pilus rod.     

Recombinant production and biochemical characterization of a hyperthermostable α-glucan/maltodextrin phosphorylase from Pyrococcusfuriosus

Alpha-glucan phosphorylase catalyzes the reversible cleavage of α-1-4-linked glucose polymers into α-D-glucose-1-phosphate. We report the recombinant production of an α-glucan/maltodextrin phosphorylase (PF1535) from a hyperthermophilic archaeon, Pyrococcus furiosus, and the first detailed biochemical characterization of this enzyme from any archaeal source using a mass-spectrometry-based assay. The apparent 98 kDa recombinant enzyme was active over a broad range of temperatures and pH, with optimal activity at 80 °C and pH 6.5–7. This archaeal protein retained its complete activity after 24 h at 80 °C in Tris-HCl buffer. Unlike other previously reported phosphorylases, the Ni-affinity column purified enzyme showed broad substrate specificity in both the synthesis and degradation of maltooligosaccharides. In the synthetic direction of the enzymatic reaction, the lowest oligosaccharide required for the chain elongation was maltose. In the degradative direction, the archaeal enzyme can produce glucose-1-phosphate from maltotriose or longer maltooligosaccharides including both glycogen and starch. The specific activity of the enzyme at 80 °C in the presence of 10 mM maltoheptaose and at 10 mg ml^–1 glycogen concentration was 52 U mg^–1 and 31 U mg^–1, respectively. The apparent Michaelis constant and maximum velocity for inorganic phosphate were 31 ± 2 mM and 0.60 ± 0.02 mM min^–1 µg^–1, respectively. An initial velocity study of the enzymatic reaction indicated a sequential bi-bi catalytic mechanism. Unlike the more widely studied mammalian glycogen phosphorylase, the Pyrococcus enzyme is active in the absence of added AMP.     

Solution structure and activation mechanism of ubiquitin-like small archaeal modifier proteins

In archaea, two ubiquitin-like small archaeal modifier protein (SAMPs) were recently shown to be conjugated to proteins in vivo. SAMPs display homology to bacterial MoaD sulfur transfer proteins and eukaryotic ubiquitin-like proteins, and they share with them the conserved C-terminal glycine-glycine motif. Here, we report the solution structure of SAMP1 from Methanosarcina acetivorans and the activation of SAMPs by an archaeal protein with homology to eukaryotic E1 enzymes. Our results show that SAMP1 possesses a β-grasp fold and that its hydrophobic and electrostatic surface features are similar to those of MoaD. M. acetivorans SAMP1 exhibits an extensive flexible surface loop between helix-2 and the third strand of the β-sheet, which contributes to an elongated surface groove that is not observed in bacterial ubiquitin homologues and many other SAMPs. We provide in vitro biochemical evidence that SAMPs are activated in an ATP-dependent manner by an E1-like enzyme that we have termed E1-like SAMP activator (ELSA). We show that activation occurs by formation of a mixed anhydride (adenylate) at the SAMP C-terminus and is detectable by SDS-PAGE and electrospray ionization mass spectrometry.     

The Structure of Physarum polycephalum hemagglutinin I suggests a minimal carbohydrate recognition domain of legume lectin fold

Physarum polycephalum hemagglutinin I (HA1) is a 104-residue protein that is secreted to extracellular space. The crystal structure of HA1 has a β-sandwich fold found among lectin structures, such as legume lectins and galectins. Interestingly, the β-sandwich of HA1 lacks a jelly roll motif and is essentially composed of two simple up-and-down β-sheets. This up-and-down β-sheet motif is well conserved in other legume lectin-like proteins derived from animals, plants, bacteria, and viruses. It is more noteworthy that the up-and-down β-sheet motif includes many residues that make contact with the target carbohydrates. Our NMR data demonstrate that HA1 lacking a jelly roll motif also binds to its target glycopeptide. Taken together, these data show that the up-and-down β-sheet motif provides a fundamental scaffold for the binding of legume lectin-like proteins to the target carbohydrates, and the structure of HA1 suggests a minimal carbohydrate recognition domain.     

Structure of the Alkalohyperthermophilic Archaeoglobus fulgidus Lipase Contains a Unique C-Terminal Domain Essential for Long-Chain Substrate Binding

Several crystal structures of AFL, a novel lipase from the archaeon Archaeoglobus fulgidus, complexed with various ligands, have been determined at about 1.8 Å resolution. This enzyme has optimal activity in the temperature range of 70-90 °C and pH 10-11. AFL consists of an N-terminal α/β-hydrolase fold domain, a small lid domain, and a C-terminal β-barrel domain. The N-terminal catalytic domain consists of a 6-stranded β-sheet flanked by seven α-helices, four on one side and three on the other side. The C-terminal lipid binding domain consists of a β-sheet of 14 strands and a substrate covering motif on top of the highly hydrophobic substrate binding site. The catalytic triad residues (Ser136, Asp163, and His210) and the residues forming the oxyanion hole (Leu31 and Met137) are in positions similar to those of other lipases. Long-chain lipid is located across the two domains in the AFL-substrate complex. Structural comparison of the catalytic domain of AFL with a homologous lipase from Bacillus subtilis reveals an opposite substrate binding orientation in the two enzymes. AFL has a higher preference toward long-chain substrates whose binding site is provided by a hydrophobic tunnel in the C-terminal domain. The unusually large interacting surface area between the two domains may contribute to thermostability of the enzyme. Two amino acids, Asp61 and Lys101, are identified as hinge residues regulating movement of the lid domain. The hydrogen-bonding pattern associated with these two residues is pH dependent, which may account for the optimal enzyme activity at high pH. Further engineering of this novel lipase with high temperature and alkaline stability will find its use in industrial applications.     

Aβ(1-40) Forms Five Distinct Amyloid Structures whose β-Sheet Contents and Fibril Stabilities Are Correlated

The ability of a single polypeptide sequence to grow into multiple stable amyloid fibrils sets these aggregates apart from most native globular proteins. The existence of multiple amyloid forms is the basis for strain effects in yeast prion biology, and might contribute to variations in Alzheimer's disease pathology. However, the structural basis for amyloid polymorphism is poorly understood. We report here five structurally distinct fibrillar aggregates of the Alzheimer's plaque peptide Aβ(1-40), as well as a non-fibrillar aggregate induced by Zn²⁺. Each of these conformational forms exhibits a unique profile of physical properties, and all the fibrillar forms breed true in elongation reactions under a common set of growth conditions. Consistent with their defining cross-β structure, we find that in this series the amyloid fibrils containing more extensive β-sheet exhibit greater stability. At the same time, side chain packing outside of the β-sheet regions contributes to stability, and to differences of stability between polymorphic forms. Stability comparison is facilitated by the unique feature that the free energy of the monomer (equivalent to the unfolded state in a protein folding reaction) does not vary, and hence can be ignored, in the comparison of ΔG° of elongation values for each polymorphic fibril obtained under a single set of conditions.     

Solution Structure of RCL, a Novel 2′-Deoxyribonucleoside 5′-Monophosphate N-glycosidase

RCL is an enzyme that catalyzes the N-glycosidic bond cleavage of purine 2′-deoxyribonucleoside 5′-monophosphates, a novel enzymatic reaction reported only recently. In this work, we determined the solution structure by multidimensional NMR and provide a structural framework to elucidate its mechanism with computational simulation. RCL is a symmetric homodimer, with each monomer consisting of a five-stranded parallel β-sheet sandwiched between five α-helices. Three of the helices form the dimer interface, allowing two monomers to pack side by side. The overall architecture featuring a Rossmann fold is topologically similar to that of deoxyribosyltransferases, with major differences observed in the putative substrate binding pocket and the C-terminal tail. The latter is seemingly flexible and projecting away from the core structure in RCL, but loops back and is positioned at the bottom of the neighboring active site in the transferases. This difference may bear functional implications in the context of nucleobase recognition involving the C-terminal carboxyl group, which is only required in the reverse reaction by the transferases. It was also noticed that residues around the putative active site show significant conformational variation, suggesting that protein dynamics may play an important role in the enzymatic function of apo-RCL. Overall, the work provides invaluable insight into the mechanism of a novel N-glycosidase from the structural point of view, which in turn will allow rational anti-tumor and anti-angiogenesis drug design.     

Allosteric signaling in the biotin repressor occurs via local folding coupled to global dampening of protein dynamics

The biotin repressor is an allosterically regulated, site-specific DNA-binding protein. Binding of the small ligand bio-5′-AMP activates repressor dimerization, which is a prerequisite to DNA binding. Multiple disorder-to-order transitions, some of which are known to be important for the functional allosteric response, occur in the vicinity of the ligand-binding site concomitant with effector binding to the repressor monomer. In this work, the extent to which these local changes are coupled to additional changes in the structure/dynamics of the repressor was investigated using hydrogen/deuterium exchange coupled to mass spectrometry. Measurements were performed on the apo-protein and on complexes of the protein bound to four different effectors that elicit a range of thermodynamic responses in the repressor. Global exchange measurements indicate that binding of any effector to the intact protein is accompanied by protection from exchange. Mass spectrometric analysis of pepsin-cleavage products generated from the exchanged complexes reveals that the protection is distributed throughout the protein. Furthermore, the magnitude of the level of protection in each peptide from hydrogen/deuterium exchange correlates with the magnitude of the functional allosteric response elicited by a ligand. These results indicate that local structural changes in the binding site that occur concomitant with effector binding nucleate global dampening of dynamics. Moreover, the magnitude of dampening of repressor dynamics tracks with the magnitude of the functional response to effector binding.