Tandem use of X‐ray crystallography and mass spectrometry to obtain ab initio the complete and exact amino acids sequence of HPBP, a human 38‐kDa apolipoprotein

Abstract

The Human Phosphate Binding Protein (HPBP) is a serendipitously discovered apolipoprotein from human plasma that binds phosphate. Amino acid sequence relates HPBP to an intriguing protein family that seems ubiquitous in eukaryotes. These proteins, named DING according to the sequence of their four conserved N-terminal residues, are systematically absent from eukaryotic genome databases. As a consequence, HPBP amino acids sequence had to be first assigned from the electronic density map. Then, an original approach combining X-ray crystallography and mass spectrometry provides the complete and a priori exact sequence of the 38-kDa HPBP. This first complete sequence of a eukaryotic DING protein will be helpful to study HPBP and the entire DING protein family.

The DING family of proteins: ubiquitous in eukaryotes, but where are the genes?

“…(6,16) As a consequence, the overall protein conformation changes upon ligand binding, a mechanism reminiscent of the Venus flytrap. (17) Crystallization and structure determination of two DING proteins, human phosphate-binding protein (HPBP) (4) and Pseudomonas fluorescens SBW25 protein (12,13) has confirmed the structural homology with bacterial PstS and the ability to bind phosphate with high affinity. (12)(13)(14)18) The conserved amino acid residues responsible for phosphate binding in PstS are present in these two proteins as well as in all DING proteins for which sequence information is available.…”

“…Extensive information is available from peptide sequencing for three DING proteins purified from eukaryotes. (3)(4)(5) Comparisons with predicted bacterial DING proteins indicate that they share around 70% identity with those. (1) Bacterial PstS, low Mr alkaline phosphatases and DING protein genes code for proteins in the expected 32-40 kDa range possessing a putative signal sequence, targeting for periplasmic localization or secretion, and all eight amino acid residues involved in phosphate binding.…”

Structure–function relationships in a bacterial DING protein

“…Despite much effort, no complete gene sequences are known for these proteins, and only a single complete amino acid sequence. The latter was determined from the X-ray crystallographic structure determination of a human phosphate-binding protein (HPBP), purified from the highdensity lipoprotein fraction of plasma, and confirmed by mass spectrometric peptide sequencing [3][4][5][6].…”

For whom the bell tolls? DING proteins in health and disease

“…The high sequence identity between DING proteins leads to the assumption that their structures are very similar, which is confirmed by the available three-dimensional models of PfluDING [5,21] and the HPBP [22][23][24]. Indeed, both DING structures possess an elongated fold composed of two adjacent globular domains (Fig.…”

“…a Cartoon representation of the structure of a DING protein representative: HPBP solved at 1.9 Å[23]. HPBP possess an elongated fold composed of two adjacent globular domains (in dark blue and cyan).…”