Crystallization and Preliminary Analysis of Crystals of the 24-Meric Hemocyanin of the Emperor Scorpion (Pandinus imperator)

Hemocyanins are giant oxygen transport proteins found in the hemolymph of several invertebrate phyla. They constitute giant multimeric molecules whose size range up to that of cell organelles such as ribosomes or even small viruses. Oxygen is reversibly bound by hemocyanins at binuclear copper centers. Subunit interactions within the multisubunit hemocyanin complex lead to diverse allosteric effects such as the highest cooperativity for oxygen binding found in nature. Crystal structures of a native hemocyanin oligomer larger than a hexameric substructure have not been published until now. We report for the first time growth and preliminary analysis of crystals of the 24-meric hemocyanin (MW = 1.8 MDa) of emperor scorpion (Pandinus imperator), which diffract to a resolution of 6.5 Å. The crystals are monoclinc with space group C 1 2 1 and cell dimensions a = 311.61 Å, b = 246.58 Å and c = 251.10 Å (α = 90.00°, β = 90.02°, γ = 90.00°). The asymmetric unit contains one molecule of the 24-meric hemocyanin and the solvent content of the crystals is 56%. A preliminary analysis of the hemocyanin structure reveals that emperor scorpion hemocyanin crystallizes in the same oxygenated conformation, which is also present in solution as previously shown by cryo-EM reconstruction and small angle x-ray scattering experiments

Die Kristallstruktur zweier Atmungsproteine: das Hämoglobin des Meerschweinchens und das 24-mere Hämocyanin des Kaiserskorpions

Im Verlauf der vorgestellten Arbeit konnten die Kristallstrukturen zweier Atmungsproteine gelöst werden. Da diese Strukturen mit verschiedenen Auflösungen gelöst wurden, wurden für die beiden Modelle unterschiedliche Methoden verwendet.rnrnDie Kristalle des Hämoglobins des Meerschweinchens Cavia porcellus erlaubten eine Messung von Streureflexen bis zu einer Auflösung von 1. 7 Ǻ. Damit konnten das Molecular Replacement und das folgende Refinement mit geringen Vorgaben durchgeführt werden.rnAnhand der ermittelten Struktur konnte gezeigt werden, dass die Höhenadaptation des Hämoglobins des Meerschweinchens auf einer Stabilisierung des R2-Zustandes und einer gleichzeitigen Destabilisierung des T-Zustandes beruht. Die zu Grunde liegenden Aminosäureaustausche konnten identifiziert und der resultierende Mechanismus postuliert werden. Die Durchführung von Mutagenese-Experimenten am Hämoglobin des Meerschweinchens könnte die vorgestellte Hypothese bestätigen.rnrnDie Kristallstruktur des 24-meren Hämocyanins aus Pandinus imperator konnte durch eine Kombination von Röntgenkristallographie und Homologiemodellierung mit einer Auflösung von 6.5 Ǻ gelöst werden. Allerdings wäre die Bestimmung der Phasen durch Molecular Replacement ohne die vor kurzem publizierte Kryo-EM Struktur nicht möglich gewesen [Cong et al., 2009].rnDamit ist es durch die Kombination verschiedener Methoden erstmalig gelungen die Struktur eines Hämocyanins dieser Größe (Mw = 1.7 MDa) zu lösen. Die Auflösung von 6.5 Ǻ, eine für die Kristallographie relativ geringe Auflösung, erlaubte die Bestimmung des Cα-Traces des Proteins mit hoher Genauigkeit.rnDurch die Kristallstruktur konnte das zu Grunde liegende Kryo-EM Modell aus [Cong et al., 2009] bestätigt werden. Insbesondere die Position der α-Helices konnte mittels Kristallographie mit einer höheren Genauigkeit bestimmt werden. Durch die Verwendung von OMIT-Maps wurde sichergestellt, dass die Struktur nicht "kopiert" wurde. Die Übereinstimmung ist deshalb auf die Struktur des Hämocyanins im gemessenen Kristall zurückzuführen, nicht auf einen Bias bei der Auswertung.rnAnhand der Kristallstruktur konnten für die Kooperativität im Trimer potentiell entscheidende Aminosäuren identifiziert werden. Diese Aminosäuren könnten im Vergleich zur bekannten trimeren Struktur aus Limulus polyphemus zur Ausbildung zusätzlicher Bindungen zwischen den Untereinheiten führen. Diese Bindungen könnten eine wichtige Rolle beim Konformationsübergang zwischen dem T- und R-Zustand spielen.rnThe crystal structures of two respiratory proteins were solved at different levels of resolutions. Therefore different methods had to be used in the modeling process.rnThe crystals of the guinea pig hemoglobin allowed diffraction measurements up to a resolution of 1.7 Ǻ. The following Molecular Replacement and Refinement could be accomplished with only low restraints, due to this high resolution.rnWith the crystal structure being solved, we were able to show that the altitude adaptation of the guinea pig hemoglobin can be explained by a stabilization of the R2-state and a simultaneous destabilization of the R-state. The underlying amino acid substitutions were identified and the resulting mechanism postulated. The conduction of mutagenesis experiments could be used to validate the presented hypothesis.rnThe crystal structure of the 24-meric hemocyanin of Pandinus imperator has been solved up to a resolution of 6.5 Ǻ by a combination of X-ray crystallography and homology modeling. The phase determination with Molecular Replacement was made possible by a recently published cryo-EM structure [Cong et al., 2009].rnThe combination of different methods therefore enabled us to solve the crystal structure of a hemocyanin of this size (Mw = 1.7 MDa) for the first time. The resolution of 6.5 Ǻ, although low for crystallography, allowed the accurate determination of the proteins Cα-Trace. rnThe underlying cryo-EM model ([Cong et al., 2009]) was confirmed by the presented crystal structure. Explicit improvement of the structure has been achieved, especially the positions of the α-helices show the higher accuracy of the crystal structure. A potential bias due to a priori information during model building was excluded by the use of OMIT-maps. rnThe crystal structure allowed the identification of amino acids that are potentially decisive for the cooperativity in the trimers of the hemocyanin. These amino acids could lead to the formation of bonds between the subunits, which have not been described in the structure of Limulus polyphemus and could play an important role in the conformational change from the T- to R-state. r

Structure of the Altitude Adapted Hemoglobin of Guinea Pig in the R2-State

Background: Guinea pigs are considered to be genetically adapted to a high altitude environment based on the consistent finding of a high oxygen affinity of their blood. Methodology/Principal Findings: The crystal structure of guinea pig hemoglobin at 1.8 A ˚ resolution suggests that the increased oxygen affinity of guinea pig hemoglobin can be explained by two factors, namely a decreased stability of the T-state and an increased stability of the R2-state. The destabilization of the T-state can be related to the substitution of a highly conserved proline (P44) to histidine (H44) in the a-subunit, which causes a steric hindrance with H97 of the b-subunit in the switch region. The stabilization of the R2-state is caused by two additional salt bridges at the b1/b2 interface. Conclusions/Significance: Both factors together are supposed to serve to shift the equilibrium between the conformational states towards the high affinity relaxed states resulting in an increased oxygen affinity

Crystallization of the altitude adapted hemoglobin of guinea pig

Hemoglobin is the versatile oxygen carrier in the blood of vertebrates and a key factor for adaptation to live in high altitudes. Several structural changes are known to account for increased oxygen affinity in hemoglobin of altitude adapted animals such as llama and barheaded goose. Guinea pigs are adapted to live in high altitudes in the Andes and consequently their hemoglobin has an increased oxygen affinity. However, the structural changes responsible for the adaptation of guinea pig hemoglobin are unknown. Here we report the crystallization of guinea pig hemoglobin in the presence of 2.6 M ammonium sulfate and a preliminary analysis of the crystals. Crystals diffract up to a resolution of 2.0 A. They are orthorhombic with space group C 2 2 2(1) and cell dimensions a = 84.08 A, b = 90.21 A and c = 83.44 A

Crystallographic parameters.

<p><b>Data collection and refinement statistics</b>.</p><p>Numbers in parentheses refer to the highest resolution shell.</p><p>*Test set size was 5% of reflections.</p>†<p><i><i>/<σ></i>  =  ratio between the mean intensity and the mean error of the intensity.</i></p

Stabilizing salt bridges of the β1/β2 interface in guinea pig hemoglobin.

<p>The β1/β2-interface of guinea pig hemoglobin in the R2-state is stabilized by two salt bridges between the N-terminal amino group of Val1 the β1-subunit and the C-terminal carboxyl group of the β2-subunit and vice versa. Both salt bridges are not present in the R2-state of human hemoglobin (1BBB)<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012389#pone.0012389-Silva1" target="_blank">[10]</a>. Guinea pig hemoglobin (β1 = red, β2 = light red) and human hemoglobin (β1 = blue, β2 = light blue) in the R2-state (PDB-code: 1BBB) were superimposed according to their C_α-atoms. Carbon atoms of the N- and C-terminal amino acids of guinea pig hemoglobin are colored light red, while carbon atoms are colored blue in human hemoglobin. Oxygen and nitrogen atoms are colored red and blue respectively. Salt bridges are denoted by dotted lines.</p

Conformational state of guinea pig hemoglobin.

<p>The structure of guinea pig hemoglobin (PDB-code: 3HYU) was superimposed with the structures of three conformational states of human hemoglobin by their C_α-atoms. The guinea pig hemoglobin structure is shown in cartoon representation, which is colored according to the distance between corresponding C_α-atoms in guinea pig hemoglobin and the respective conformational state of human hemoglobin in (A) T-state (PDB-code: 1A3N, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012389#pone.0012389-Tame1" target="_blank">[36]</a>), (B) R-state (PDB-code: 1HHO, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012389#pone.0012389-Shaanan1" target="_blank">[37]</a>) and (C) R2-state (PDB-code: 1BBB, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012389#pone.0012389-Silva1" target="_blank">[10]</a>). Distances between C_α-atoms clearly show that guinea pig hemoglobin crystallizes in the R2-state (C). Color coding of C_α-atoms distances was made according to the colors given in the bar below.</p

The “Switch” region of the α1/β2 interface in guinea pig hemoglobin.

<p>The switch region at the α1/β2 interface in guinea pig hemoglobin shows two important differences in comparison with human hemoglobin in the R2-state. Firstly, the stabilizing salt bridge, which connects Glu30 and His50 in human hemoglobin, is missing due to an amino acid exchange in guinea pig hemoglobin. Secondly, a steric hindrance between His97 of the β2-subunit and His44 of the α1-subunit might render the T-state of guinea pig hemoglobin less stable than the T-state in human hemoglobin, which has a Pro44 in the α1-subunit. Due to this steric hindrance a relaxed state conformation (R- or R2-state) of guinea pig hemoglobin could be favored, thereby increasing its oxygen affinity. The α1- and β2-subunit of guinea pig hemoglobin (α1 = light red) and human hemoglobin (α1 = light blue) in the R2-state (PDB-code: 1BBB, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012389#pone.0012389-Silva1" target="_blank">[10]</a>) were superimposed according to their C_α-atoms. Carbon atoms of amino acids in guinea pig hemoglobin are colored light red, while the carbon atoms are colored blue in human hemoglobin. The position of the β2-subunit is denoted by a light grey area, while the sliding movement of His97 in the course of the conformational transition is illustrated by an arrow (dark grey). The position of His97 in the R2-state of guinea pig hemoglobin is colored in light red. Furthermore the position of His97 in human hemoglobin is shown in the T-state (grey, PDB-code: 1A3N, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012389#pone.0012389-Tame1" target="_blank">[36]</a>), R-state (orange, PDB-code: 1HHO, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012389#pone.0012389-Shaanan1" target="_blank">[37]</a>) and R2-state (light blue, PDB-code: 1BBB, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012389#pone.0012389-Silva1" target="_blank">[10]</a>).</p

Crystallographic parameters.

<p>Crystallographic parameters.</p