The Allosteric Effector l-Lactate Induces a Conformational Change of 2×6-meric Lobster Hemocyanin in the Oxy State as Revealed by Small Angle X-ray Scattering

Abstract

Hemocyanins are multisubunit respiratory proteins found in many invertebrates. They bind oxygen highly cooperatively. However, not much is known about the structural basis of this behavior. We studied the influence of the physiological allosteric effector L-lactate on the oxygenated quaternary structure of the 2؋6-meric hemocyanin from the lobster Homarus americanus employing small angle x-ray scattering (SAXS). The presence of 20 mM L-lactate resulted in different scattering curves compared with those obtained in the absence of L-lactate. The distance distribution functions p(r) indicated a more compact molecule in presence of L-lactate, which is also reflected in a reduction of the radius of gyration by about 0.2 nm (3%). Thus, we show for the first time on a structural basis that a hemocyanin in the oxy state can adopt two different conformations. This is as predicted from the analysis of oxygen binding curves according to the "nesting" model. A comparison of the distance distribution functions p(r) obtained from SAXS with those deduced from electron microscopy revealed large differences. The distance between the two hexamers as deduced from electron microscopy has to be shortened by up to 1.1 nm to agree well with the small angle x-ray curves.Hemocyanins are the oxygen transport proteins of most molluscs and arthropods. They are enormous molecular structures, ranging in molecular mass from 4.5 ϫ 10 5 to more than 10 7 Da; some contain more than 100 oxygen binding sites (1-3). Hemocyanins exhibit cooperative oxygen binding and respond to a variety of allosteric effectors (4 -8). As a consequence of their structural complexity, hemocyanins have proved important in extending our understanding of allostery. For example, explanation of the cooperative oxygen binding by arthropod hemocyanin has required extension of the classical MWC 1 model to the "nesting" model (8, 9), which reflects the hierarchical structure of these proteins. The nesting model requires four states (rT, rT, tR, rR) instead of the two states (T, R) of the MWC model. It makes the prediction that even fully oxygenated hemocyanin can exist in two conformational states. Although the model has served well to explain oxygen binding and its dependence on effector (see Ref. 8, for example), it has never been tested directly. That is, no experiments have been performed to detect the two oxy conformations predicted by the nesting model. To be definitive, such experiments must be carried out in solution, under rigorously controlled oxygenation levels and with a sensitivity sufficient to detect even small conformational changes.A suitable method to detect conformational changes of large proteins in solution is small angle x-ray scattering (SAXS). This technique has been applied successfully to monitor changes of the quaternary structure for several cooperative proteins such as hemoglobin and a number of allosteric enzymes (10 -18). Different conformations have also been reported for the oxy-and deoxyhemocyanin from the tarantula Eurypelma californicum fro...

Solution structures of 2 × 6-meric and 4 × 6-meric hemocyanins of crustaceans Carcinus aestuarii, Squilla mantis and Upogebia pusilla

“…1A and B and the obtained invariants radius of gyration (R g ) and maximum particle length (D max ) are reported in Table 1. R g and D max are 72 ± 1 and 225 ± 5 Å, respectively, in very good agreement with those reported for the 2 Â 6-meric Hc from H. americanus (72.1 ± 0.05 and 235 Å, respectively (Hartmann et al, 2001). The molecular mass determined from the forward scattering intensity is in agreement with the 2 Â 6-meric state of this Hc species (Table 1).…”

“…With these approaches, the quaternary structures of the 1 Â 6-meric Hc from lobsters Palinurus elephas (by cryo-EM, Meissner et al, 2003) and Homarus americanus (by SAXS, Hartmann et al, 2001), horseshoe crab L. polyphemus, centipede Scutigera coleoptrata and scorpion Pandinus imperator (by cryo-EM, Taveau et al, 1997;Martin and et al, 2007;Markl et al, 2009;Cong and et al, 2009) and the tarantula Eurypelma californicum (by SAXS and EM, Hartmann and Decker, 2002;Decker et al, 1996;De Haas and van Bruggen, 1994) have been solved to a resolution as good as $20-30 Å in case of SAXS and $7 Å in case of cryo-EM.…”

Cooperative Transition in the Conformation of 24-Mer Tarantula Hemocyanin upon Oxygen Binding

“…Typical is the dominance of the state with the lowest oxygen affinity (tT) in the absence of oxygen, against which conformations with high oxygen affinities (rR, rT) are predominant in the presence of oxygen. The persistence of two states at high oxygen concentrations is due to a limited allosteric range and experimentally confirmed for arthropod hemocyanins (29). DISCUSSION The hemocyanin of tarantula E. californicum shows highly cooperative oxygen binding, possess 24 oxygen-binding sites, and is able to adopt different conformations (10,30,31).…”

Structural Basis of the Lactate-dependent Allosteric Regulation of Oxygen Binding in Arthropod Hemocyanin

“…Oxygen binding by Hcs is finely regulated by a number of allosteric effectors such as H ϩ (9, 13), L-lactate (10), and urate (14). Several models (two-state, three-state, and nested Monod-Wyman-Changeux models) have been applied to describe the binding affinity and cooperativity of arthropod Hcs and their dependence on solution conditions (15).…”

“…Hcs bind oxygen cooperatively, and as a general rule, the more complex quaternary structures with higher molecular mass exhibit stronger cooperative interactions manifested in increased Hill coefficients. Investigation of the origins of cooperativity has been approached through examination of the three-dimensional crystal structures of deoxy-and oxy-Hc (8) as well as through x-ray absorption spectroscopy (XAS) (9) and small angle x-ray scattering (SAXS) (10,11), in some cases in the presence of small molecules acting as allosteric modifiers and/or at low pH. The results of these studies indicate conformational changes that occur at the level of the active site as well as the tertiary and quaternary structure of multimers.…”