Structural investigation of a phosphorylation-catalyzed, isoaspartate-free, protein succinimide: crystallographic structure of post-succinimide His15Asp histidine-containing protein

Abstract

Aspartates and asparagines can spontaneously cyclize with neighboring main-chain amides to form succinimides. These succinimides hydrolyze to a mixture of isoaspartate and aspartate products. Phosphorylation of aspartates is a common mechanism of protein regulation and increases the propensity for succinimide formation. Although typically regarded as a form of protein damage, we hypothesize succinimides could represent an effective mechanism of phosphoaspartate autophosphatase activity, provided hydrolysis is limited to aspartate products. We previously reported the serendipitous creation of a protein, His15Asp histidine-containing protein (HPr), which undergoes phosphorylation-catalyzed formation of a succinimide whose hydrolysis is seemingly exclusive for aspartate formation. Here, through the high-resolution structure of postsuccinimide His15Asp HPr, we confirm the absence of isoaspartate residues and propose mechanisms for phosphorylation-catalyzed succinimide formation and its directed hydrolysis to aspartate. His15Asp HPr represents the first characterized protein example of an isoaspartate-free succinimide and lends credence to the hypothesis that intramolecular cyclization could represent a physiological mechanism of autophosphatase activity. Furthermore, this indicates that current strategies for succinimide evaluation, based on isoaspartate detection, underestimate the frequencies of these reactions. This is considerably significant for evaluation of protein stability and integrity.

FIGURE 1

Mechanism of succinimide and isoimide formation and hydrolysis. (A) Succinimide formation and hydrolysis. Mechanism of succinimide formation and hydrolysis from an asparagine and a phosphoaspartate. A similar mechanism occurs from aspartate, with the distinction being release of a water molecule rather than a phosphoryl group upon ring formation. Also shown are the products which emerge from succinimide hydrolysis with aspartates being produced from hydrolysis at the side-chain carbonyl and isoaspartates from hydrolysis at the main-chain carbonyl. (B) Isoimide formation and hydrolysis. Hydrolysis of an isoimide ring results in aspartyl products independent of the point of attack of the incoming water molecule.

FIGURE 2

Phosphorylation of His15Asp generates three species of the protein. His15Asp HPr (3 mg) was phosphorylated at 37 °C for 60 min in 10 mM potassium phosphate buffer (pH 7.0) with 5 mM phosphoenolpyruvate, 5 mM MgCl₂, and 0.3 mg of enzyme I. Protein species were separated by isoelectric focusing: lane 1, unphosphorylated His15Asp HPr; lane 2, His15Asp HPr under phosphorylating conditions with the generation of both the phosphorylated and high-pI species; lane 3, isolated high-pI His15Asp HPr; and lane 4, isolated high-pI His15Asp HPr dialyzed overnight against PBS.

FIGURE 3

Regions of structural deviation between His15Asp HPr and PS-His15Asp HPr. Root-mean-square deviations were calculated for the α-carbons using the LSQKAB program from CCP4: (A) rmsds of the α-carbons of the two molecules within the asymmetric unit of PS-His15Asp HPr and (B) rmsds of the α-carbons of pre- and post-succinimide PS-His15Asp HPr.

FIGURE 4

Structural deviation between His15Asp and PS-His15Asp HPr. These structures were created with Pymol (30), and in each case, molecules with green carbon–carbon bonds represent the PS-His15Asp HPr whereas molecules with blue carbon–carbon bonds represent His15Asp HPr. Structural comparisons are presented for the following. (A) Minor helix B. Formation of a more idealized helix in His15Asp Hpr is believed to be a consequence of a sulfate anion from the crystallization solution. (B) C-Terminus. The similar positioning of this region in two different crystallographic investigations of His15Asp HPr supports the proposed role in preventing isoaspartyl formation. (C) Active center. This involves a general repositioning of the main chain to bring it closer to the core of the protein as well as a tightening of the cleft that contains the active site region.

FIGURE 5

Stereographic representation of electron density of th active site region. Electron density of the active site region of post-succinimide His15Asp HPr at 1 Å resolution. The clear electron density for Asp15 as well as the absence of additional electron density that is unaccounted for in the active center region argues against the possibility of an isoaspartyl residue at position 15.

FIGURE 6

Amino acid analysis and methyltransferase assays for detection of isoaspartyls in PS-His15Asp HPr. (A) Amino acid sequencing. The inability for N-terminal sequencing to proceed past nonstandard peptide bonds, such as succinimides or isoaspartyls, provides a qualitative assessment for the presence of these residues. The inability to sequence past the 14th residue of high-pI His15Asp HPr indicates the presence of a succinimide, while detection of correct sequences in positions 15 and 16 of PS-His15Asp HPr indicates breakdown to an aspartyl product. Not shown are the first 10 residues of each protein. (B) Methyltransferase assays were performed using the ISOQUANT isoaspartate kit (Promega) according to the manufacturer’s instructions. Isoaspartate content is expressed as a percentage of modified residues per 100 molecules of protein. Isoaspartate contents were determined for the wild type, His15Asp HPr, and PS-His15Asp HPr for both intact and proteolytic fragments, to eliminate any influence of protein structure on the ability to act as a PIMT substrate. Each data point represents separate PIMT assay.

FIGURE 7

Stereographic comparison of the phosphoacceptor site of the wild type, His15Asp HPr, and PS-His15Asp HPr. This figure was created with Pymol (30). Molecules with green carbon–carbon bonds represent the post-succinimide structure, molecules with blue carbon–carbon bonds the pre-succinimide structure, and molecules with magenta carbon–carbon bonds wild-type HPr. For both the preand post-succinimide His15Asp structures, the carboxyl group oxygen occupies a position similar to that of the N^δ1 atom of the phosphoacceptor His15 residue of wild-type HPr.

FIGURE 8

Structural mechanism for succinimide-based autophosphatase activity. (A) Structural isomers of phosphorylation and succinimide-based dephosphorylation of His15Asp are modeled on the basis of the crystallographic structure. (B) General mechanism for catalyzed succinimide formation from a phosphoaspartate with subsequent hydrolysis to aspartate.