The Length of the A-M3 Linker Is a Crucial Determinant of the Rate of the Ca2+ Transport Cycle of Sarcoplasmic Reticulum Ca2+-ATPase

Ion translocation by the sarcoplasmic reticulum Ca²⁺-ATPase depends on large movements of the A-domain, but the driving forces have yet to be defined. The A-domain is connected to the ion-binding membranous part of the protein through linker regions. We have determined the functional consequences of changing the length of the linker between the A-domain and transmembrane helix M3 (“A-M3 linker”) by insertion and deletion mutagenesis at two sites. It was feasible to insert as many as 41 residues (polyglycine and glycine-proline loops) in the flexible region of the linker without loss of the ability to react with Ca²⁺ and ATP and to form the phosphorylated Ca₂E1P intermediate, but the rate of the energy-transducing conformational transition to E2P was reduced by >80%. Insertion of a smaller number of residues gave effects gradually increasing with the length of the insertion. Deletion of two residues at the same site, but not replacement with glycine, gave a similar reduction as the longest insertion. Insertion of one or three residues in another part of the A-M3 linker that forms an α-helix (“A3 helix”) in E2/E2P conformations had even more profound effects on the ability of the enzyme to form E2P. These results demonstrate the importance of the length of the A-M3 linker and of the position and integrity of the A3 helix for stabilization of E2P and suggest that, during the normal enzyme cycle, strain of the A-M3 linker could contribute to destabilize the Ca₂E1P state and thereby to drive the transition to E2P.The sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA)² is a membrane-bound ion pump that transports Ca²⁺ against a steep concentration gradient, utilizing the energy derived from ATP hydrolysis (1–3). It belongs to the family of P-type ATPases, in which the γ-phosphoryl group of ATP is transferred to a conserved aspartic acid residue during the reaction cycle. Both phospho and dephospho forms of the enzyme undergo transitions between so-called E1 and E2 conformations (Scheme 1). The E1 and E1P states display specificity for reaction with ATP and ADP, respectively (“kinase activity”), whereas E2P and E2 react with water and P_i instead of nucleotide (“phosphatase activity”). The E1 dephosphoenzyme of the Ca²⁺-ATPase binds two Ca²⁺ ions with high affinity from the cytoplasmic side, thereby triggering the phosphorylation from ATP. In E1P, the Ca²⁺ ions are occluded with no access to either side of the membrane, and Ca²⁺ is released to the luminal side after the conformational transition to E2P, likely in exchange for protons being countertransported. The structural organization and domain movements leading to Ca²⁺ translocation have recently been elucidated by crystallization of SERCA in various conformational states thought to represent intermediates in the pump cycle (4–7). SERCA is made up of 10 membrane-spanning mostly helical segments, M1–M10 (numbered from the N terminus), of which M4–M6 and M8 contribute liganding groups for Ca²⁺ binding, and a cytoplasmic headpiece separated into three distinct domains, named A (“actuator”), P (“phosphorylation”), and N (“nucleotide binding”). The A-domain appears to undergo considerable movement during the functional cycle. In the E1/E1P states, the highly conserved TGE¹⁸³S loop of the A-domain is at great distance from the catalytic center containing nucleotide-binding residues and the phosphorylated Asp³⁵¹ of the P-domain, but during the Ca₂E1P → E2P transition, the A-domain rotates ∼90° around an axis perpendicular to the membrane, thereby moving the TGE¹⁸³S loop into close contact with the catalytic site such that Glu¹⁸³ can catalyze dephosphorylation of E2P (8, 9). During the dephosphorylation, Glu¹⁸³ likely coordinates the water molecule attacking the aspartyl phosphoryl bond and withdraws a hydrogen. Hence, the movement of the A-domain during the Ca₂E1P → E2P transition is the event that changes the catalytic specificity from kinase activity to phosphatase activity. During the dephosphorylation of E2P → E2, there is only a slight change of the position of the A-domain, and a large back-rotation is needed to reach the E1 form from E2; thus, the A-domain rotation defines the difference between the E1/E1P class of conformations and the E2/E2P class. Because the A-domain is physically connected to transmembrane helices M1–M3 through the linker segments A-M1, A-M2, and A-M3, the A-domain movement occurring during the Ca₂E1P → E2P transition may be a key event in the opening of the Ca²⁺ sites toward the lumen, thus explaining the coupling of ATP hydrolysis to Ca²⁺ translocation. An important unanswered question is, however, how the movement of the A-domain is brought about. Which are the driving forces that destabilize Ca₂E1P and/or stabilize E2P such that the energy-transducing Ca₂E1P → E2P transition takes place? To answer this, it seems important to elucidate the exact roles of the linkers. Intriguing results have been obtained by Suzuki and co-workers, who demonstrated the importance of the A-M1 linker in connection with luminal release of Ca²⁺ from E2P (10). In this study, we have addressed the role of the A-M3 linker. An alignment of two crystal structures thought to resemble the Ca₂E1P and E2·P_i forms (5), respectively, is shown in Fig. 1. The A-domain rotation is associated with formation of a helix (“A3 helix”) in the N-terminal part of the A-M3 linker, and this helix seems to interact with a helix bundle consisting of the P5–P7 helices of the P-domain, a feature exhibited by all published crystal structures of the E2 type (cf. supplemental Fig. S1 and Ref. 11). Moreover, when structures of similar crystallographic resolution are compared (as in Fig. 1), the non-helical part of the A-M3 linker in E2-type structures has a higher relative temperature factor (“B-factor”) than the corresponding segment in Ca₂E1P (Fig. 1C, thick part colored orange-red for high temperature factor), thus suggesting a higher degree of freedom of movement relative to Ca₂E1P. Hence, the A-M3 linker appears more strained in Ca₂E1P compared with E2 forms, and the greater flexibility of the linker in E2 forms may promote the formation of the A3 helix.Open in a separate windowSCHEME 1.Ca²⁺-ATPase reaction cycle.Open in a separate windowFIGURE 1.A-M3 linker configuration in E1- and E2-type crystal structures. Crystal structures with Protein Data Bank codes 2zbd (Ca₂E1P analog) and 1wpg (E2·P_i analog) are shown aligned. A, overview of structure 2zbd in bluish colors with green A-M3 linker and structure 1wpg in reddish colors with wheat A-M3 linker. B, magnification of the A-M3 linker (corresponding to the red box in A) with arrows indicating site 1, between Glu²⁴³ and Gln²⁴⁴, and site 2, between Gly²³³ and Lys²³⁴, in both conformations. The green A-M3 linker to the right is structure 2zbd. The wheat A-M3 linker to the left is structure 1wpg. Note the kinked A3 helix forming part of the latter structure. C, same A-M3 linker structures as in B but with the magnitude of the temperature factor (B-factor) indicated in colors (red > orange > yellow > green > blue) and by tube diameter. Because the two crystal structures selected here as E1- and E2-type representatives have similar crystallographic resolution (2.40 and 2.30 Å, respectively), the differences in temperature factor in specific regions provide direct information about chain flexibility.Here, we have determined the functional consequences of changing the length (and thereby likely the strain) of the A-M3 linker. Polyglycine and glycine-proline loops of varying lengths were inserted at two different sites in the linker (Fig. 1), and deletions were also studied. Rather unexpectedly, we were able to insert as many as 41 residues in one of the sites without loss of expression or ability to react with Ca²⁺ and ATP, forming Ca₂E1P, but the Ca₂E1P → E2P transition was greatly affected.     

Meal and oral glucose tests for assessment of beta -cell function: modeling analysis in normal subjects

We investigated beta-cell function and its relationship to insulin sensitivity in 17 normal volunteers. For insulin secretion (derived by C-peptide deconvolution), a mathematical model was applied to 24-h triple-meal tests (MT) as well as oral glucose tolerance tests (OGTT); insulin sensitivity was assessed by the euglycemic insulin clamp technique. The beta-cell model featured a glucose concentration-insulin secretion dose response (characterized by secretion at 5 mM glucose and slope), a secretion component proportional to the glucose concentration derivative, and a time-dependent potentiation factor (modulating the dose response and accounting for effects of sustained hyperglycemia and incretins). The beta-cell dose-response functions estimated from the whole 24-h MT, the first 2 h of the MT, and the OGTT differed systematically, because a different potentiation factor was involved. In fact, potentiation was higher than average during meals (1.6 +/- 0.1-fold during the first meal) and had a different time course in the MT and OGTT. However, if potentiation was accounted for, the 24- and 2-h MT and the OGTT yielded similar dose responses, and most beta-cell function parameters were intercorrelated (r = 0.50-0.86, P < or = 0.05). The potentiation factor was found to be related to plasma glucose-dependent insulin-releasing polypeptide concentrations (r = 0.49, P < 0.0001). Among beta-cell function parameters, only insulin secretion at 5 mM glucose from MT correlated inversely with insulin sensitivity (24-h MT: r = -0.74, P < 0.001; 2-h MT: r = -0.52, P < 0.05), whereas the dose-response slope and the OGTT parameters did not. In nine other subjects, reproducibility of model parameters was evaluated from repeated MTs. Coefficients of variation were generally approximately 20%, but the derivative component was less reproducible. We conclude that our model for the multiple MT yields useful information on beta-cell function, particularly with regard to the role of potentiation. With cautious interpretation, a 2-h MT or a standard OGTT can be used as surrogates of 24-h tests in assessing spontaneous beta-cell function.     

Dual-affinity avidin molecules

A recently reported dual-chain avidin was modified further to contain two distinct, independent types of ligand-binding sites within a single polypeptide chain. Chicken avidin is normally a tetrameric glycoprotein that binds water-soluble d-biotin with extreme affinity (K(d) approximately 10(-15) M). Avidin is utilized in various applications and techniques in the life sciences and in the nanosciences. In a recent study, we described a novel avidin monomer-fusion chimera that joins two circularly permuted monomers into a single polypeptide chain. Two of these dual-chain avidins were observed to associate spontaneously to form a dimer equivalent to the wt tetramer. In the present study, we successfully used this scaffold to generate avidins in which the neighboring biotin-binding sites of dual-chain avidin exhibit two different affinities for biotin. In these novel avidins, one of the two binding sites in each polypeptide chain, the pseudodimer, is genetically modified to have lower binding affinity for biotin, whereas the remaining binding site still exhibits the high-affinity characteristic of the wt protein. The pseudotetramer (i.e., a dimer of dual-chain avidins) has two high and two lower affinity biotin-binding sites. The usefulness of these novel proteins was demonstrated by immobilizing dual-affinity avidin with its high-affinity sites. The sites with lower affinity were then used for affinity purification of a biotinylated enzyme. These "dual-affinity" avidin molecules open up wholly new possibilities in avidin-biotin technology, where they may have uses as novel bioseparation tools, carrier proteins, or nanoscale adapters.     

Design and construction of highly stable, protease-resistant chimeric avidins

The chicken avidin gene family consists of avidin and seven separate avidin-related genes (AVRs) 1-7. Avidin protein is a widely used biochemical tool, whereas the other family members have only recently been produced as recombinant proteins and characterized. In our previous study, AVR4 was found to be the most stable biotin binding protein thus far characterized (T(m) = 106.4 degrees C). In this study, we studied further the biotin-binding properties of AVR4. A decrease in the energy barrier between the biotin-bound and unbound state of AVR4 was observed when compared with that of avidin. The high resolution structure of AVR4 facilitated comparison of the structural details of avidin and AVR4. In the present study, we used the information obtained from these comparative studies to transfer the stability and functional properties of AVR4 to avidin. A chimeric avidin protein, ChiAVD, containing a 21-amino acid segment of AVR4 was found to be significantly more stable (T(m) = 96.5 degrees C) than native avidin (T(m) = 83.5 degrees C), and its biotin-binding properties resembled those of AVR4. Optimization of a crucial subunit interface of avidin by an AVR4-inspired point mutation, I117Y, significantly increased the thermostability of the avidin mutant (T(m) = 97.5 degrees C) without compromising its high biotin-binding properties. By combining these two modifications, a hyperthermostable ChiAVD(I117Y) was constructed (T(m) = 111.1 degrees C). This study provides an example of rational protein engineering in which another member of the protein family has been utilized as a source in the optimization of selected properties.     

The effect of hydrogen bonds on the conformation of glycosphingolipids. Methylated and unmethylated cerebroside studied by X-ray single crystal analysis and model calculations

The conformation and molecular packing of permethylated beta-D-galactosyl-N-octadecanoyl-D-spingosine (cerebroside) was determined by X-ray single crystal analysis at 185 K (R = 0.16). The lipid crystallizes in the orthorhombic space group P2(1)2(1)2(1) with the unit cell dimensions a = 8.03, b = 7.04 and c = 88.10 A. The four molecules in the unit cell pack in a bilayer arrangement with tilting (48 degrees) hydrocarbon chains. The direction of the chain tilt alternates in the two bilayer halves and in adjacent bilayers. In order to define the effect of hydrogen bonds on the molecular conformation the structural features of the permethylated cerebroside are compared with that of unsubstituted cerebroside (I. Pascher and S. Sundell (1977) Chem. Phys. Lipids 20, 179). It is shown that methylation of the hydrogen donor groups does not affect the conformation of the ceramide part. However, by abolishing the intramolecular hydrogen bond between the amide N--H group and the glycosidic oxygen the galactose ring changes its orientation from layer-parallel to layer-perpendicular. Calculations using molecular mechanics, MM2(87), show that in natural cerebroside the intramolecular hydrogen bond stabilizes the theta 1 = -syn-clinal conformation about the C(1)--C(2) sphingosine bond by 2-2.5 kcal/mol compared to other staggered conformations. The significance of the L shape of the native cerebroside, making both the carbohydrate and polar ceramide groups accessible as a binding epitope in recognition processes, is discussed.     

Territorial defense against various food competitors in the Tanganyikan benthophagous cichlid <Emphasis Type="Italic">Neolamprologus tetracanthus</Emphasis>

Territorial defense of nonbreeding female Neolamprologus tetracanthus, a shrimp-eating Tanganyikan cichlid, was investigated. Females defended territories (=home ranges, ca. 1m across) against a variety of intruding fishes. Conspecific females were usually attacked outside the territories, heterospecific benthivores (shrimp eaters) and omnivores near the border of the territories, and piscivores, algae and detritus feeders, and herbivores inside the territories. Females used some parts of the sandy substrate in the territories for foraging (foraging areas). Territorial defense prevented most of the conspecific females and benthivores from intruding into the foraging areas. In omnivores, piscivores, and algae and detritus feeders, about half the intruders were repelled from the foraging areas, although herbivores were infrequently repelled in the areas. Soon after removal of the resident females, many food competitors invaded the foraging areas and eagerly devoured prey, suggesting that the territories are maintained for food resource protection from these competitors. Females are likely to discriminate intruding fishes and change their territorial defense primarily on the basis of the degree of dietary overlap, resulting in monofunctional serial territories.