Crystal Structure of <em>Cryptosporidium parvum</em> Pyruvate Kinase

<div><p>Pyruvate kinase plays a critical role in cellular metabolism of glucose by serving as a major regulator of glycolysis. This tetrameric enzyme is allosterically regulated by different effector molecules, mainly phosphosugars. In response to binding of effector molecules and substrates, significant structural changes have been identified in various pyruvate kinase structures. Pyruvate kinase of <em>Cryptosporidium parvum</em> is exceptional among known enzymes of protozoan origin in that it exhibits no allosteric property in the presence of commonly known effector molecules. The crystal structure of pyruvate kinase from <em>C. parvum</em> has been solved by molecular replacement techniques and refined to 2.5 Å resolution. In the active site a glycerol molecule is located near the γ-phosphate site of ATP, and the protein structure displays a partially closed active site. However, unlike other structures where the active site is closed, the α6' helix in <em>C. parvum</em> pyruvate kinase unwinds and assumes an extended conformation. In the crystal structure a sulfate ion is found at a site that is occupied by a phosphate of the effector molecule in many pyruvate kinase structures. A new feature of the <em>C. parvum</em> pyruvate kinase structure is the presence of a disulfide bond cross-linking the two monomers in the asymmetric unit. The disulfide bond is formed between cysteine residue 26 in the short N-helix of one monomer with cysteine residue 312 in a long helix (residues 303–320) of the second monomer at the interface of these monomers. Both cysteine residues are unique to <em>C. parvum</em>, and the disulfide bond remained intact in a reduced environment. However, the significance of this bond, if any, remains unknown at this time.</p> </div

Data-collection and refinement statistics.

a<p>Values in parentheses are for the outermost resolution shell.</p

Cartoon drawing showing superposition of <i>C. parvum</i> pyruvate kinase structures.

<p>Our structure is orange, except for the B domain, which is magenta; 3MA8 is light green. Sulfate ions are shown as stick models (our structure - yellow and red; 3MA8 - green). The α6’ helix in our structure is highlighted in red.</p

Primary sequences of pyruvate kinases from various organisms were aligned using the CLUSTALW program [33], [34].

<p>The labeling of secondary structural elements corresponds to the CpPyK structure. The two black triangles indicate the cysteine residues involved in the disulfide bond. The stars mark the characteristic 6-residue insertion preceding the β5 strand in domain A of CpPyK.</p

Binding of sulfate ions in CpPyK.

<p>Potential hydrogen bond donors and acceptors are indicated by dotted lines with distances in Å. (A) SULF1 and nearby residues in the allosteric site. (B) SULF2 and nearby residues.</p

CpPyK asymmetric unit.

<p>Monomers A and B comprise the asymmetric unit and are related by a noncrystallographic 2-fold axis perpendicular to the plane of the paper. The domains in each monomer are colored as follows: N - cyan (residues 23–32), A - wheat (residues 42–112 and 212–389), B - magenta (residues 113–211), and C - light green (residues 390–526). The sulfate ions are shown as stick models; the sulfate ion bound at the effector site in each monomer is labeled SULF1. The sulfur atoms of cysteine residues 26 and 312 in each monomer are shown as orange balls; the disulfide is indicated by CC. The unwound helix α6’ is shown in red in both monomers. The A domains from these monomers form the major protein-protein interface in the tetramer.</p

Cartoon drawing of monomers A (light pink) and B (light cyan) showing the orientation of the Arg342 side chain from monomer. A.

<p>Unwinding of the α6’ helix of monomer B results in the movement of the main chain carbonyl group of Gly295 too far away for interaction with Arg342 of monomer A. Arg294, Thr328 and Gln329 of the B monomer are also shown.</p

CpPyk tetramer and monomer.

<p>(A) The tetramer is generated by a crystallographic 2-fold axis. Domains of monomer A are colored the same as in Fig. 1. Symmetry related monomers are shown in green and orange. A minor interface is formed by the C-domains of the symmetry partners. (B) Monomer A. The domains are colored as follows: N - light pink, A - orange, B - magenta, C - light green. The two sulfate ions are labeled SULF1 and SULF2. The N-helix of the B monomer (cyan) is included in order to show the disulfide bond. The sulfur atoms in the disulfide bond between cysteine residues 26 and 312 are shown as orange balls. Glycerol and acetate ions are shown as stick models. The unwound helix α6’ is shown in red. The location of the missing effector loop is indicated. The loop representing the Cryptosporidium-specific insertion in the primary sequence is also labeled.</p

Comparison of CpPyK with LmPyK.

<p>(A) Superposition of A monomers of CpPyK (red), <i>L. mexicana</i> PyK without sulfate ion in the active site (1PKL, green), and <i>L. mexicana</i> PyK with sulfate ion in the active site (3E0V, blue). Sulfate ions bound at the effector binding site (labeled E) in all three structures are shown as stick models: CpPyK (yellow and red), 1PKL (green) and 3E0V (blue). Sulfate ions in the active site area (labeled A) in 3E0V are also shown in blue. One additional sulfate in the CpPyK structure at the interface of the C and A domains is shown in magenta. The glycerol molecule in the active site of CpPyK is shown as a ball and stick model. The two areas of the protein structures affected by the insertions in <i>L. mexicana</i> and <i>C. parvum</i> sequences are labeled. (B) Unwinding of helix α6’ in both monomers of CpPyK (red and yellow) as compared to 3E0V (cyan) and 1PKL (green). Residues 293 and 302 for CpPyK are labeled.</p