Histidine triad (HIT) proteins are a superfamily of proteins named for a His-ϕ-His-ϕ-His-ϕ-ϕ motif near the C-terminus in which ϕ is a hydrophobic amino acid1
. This motif was reported be a binding site for zinc ion2
. Primary sequence alignment of HIT proteins () reveals that there are two branches. One branch contains representatives in all three domains of cellular life, the eukarya, bacteria, and archaea. The second branch appears to be confined to eukarya, with representative sequences in humans and two yeast genera.
Fig. 1 Sequence alignment of HIT proteins excluding nearly identical proteins, expressed sequence tags and partial sequences. Strict conservation of residues aligning with Phe 19, His 51, Leu 53, and Histidines 110, 112 and 114 was required for inclusion. The (more ...)
We purified HINT (histidine triad nucleotide-binding protein), a member of the first branch of the HIT superfamily, as a dimeric purine nucleoside and nucleotide-binding protein from rabbit heart cytosol (J.G., N. Liang & J.M.L, in prep.). HINT is nearly identical to proteins that have been given the designation protein kinase C inhibitor-1 (PKCI-1). Bovine PKCI-1 was so named because it was present in brain cytosol fractions that inhibited a mixture of PKC isoforms3
. It has not been possible to reproduce this inhibition with HINT from rabbit heart or with purified recombinant HINT (J.G., N. Liang & J.M.L., in prep.). The corresponding human cDNA was cloned in two independent two-hybrid screens. In the first screen, interactions were sought with the product of ATDC4
, a cDNA that when overexpressed, suppresses the radiation-sensitivity of a fibroblast cell line from a child with Ataxia-Telangiectasia. In the second screen, interactions were sought with the N-terminal 317 amino acid regulatory domain of PKC isozyme β5
. These authors refer to the protein as PKCI-1 (protein kinase C interacting
protein) on account of the yeast two-hybrid interaction. The human gene has been mapped to 5q31.26
The HINT branch of the HIT superfamily contains proteins found in all three domains of cellular life (). Homologs within the eukarya are not restricted to those in plants7
but represent, additionally, distant eukarya such as Dictyostelium discoidium
(Genbank accession number U61986). Within the contiguous core of 102 amino acids that includes all secondary structural elements of HINT, mammalian HINT proteins are nearly identical. HINT proteins from nonmammalian animals and nonanimal eukaryotes represent greater variety. The flatworm Caenorabditis elegans8
contains two HINT-related proteins, one with 68 and the other with 25 identities within the core. Homologs from Zea mays7
and Saccharomyces cerevisiae9
contain, respectively, 54 and 32 identities within this 102 amino acid region.
Remarkably, several predicted proteins from bacteria and archaea have been identified that have greater identity with mammalian HINT than do eukaryotic counterparts. The cyanobacterial Synechococcus
has a homolog10
more similar to mammalian HINT than is the Zea
protein. The proteobacterial Haemophilus11
genera and the methanogenic archaeon Methanococcus13
have HINT proteins more similar to mammalian HINT than the Saccharomyces
HINT protein, Hnt1. The smallest known genome of a free-living organism, that of Mycoplasma genitalium
, has 470 predicted coding regions. It encodes a HINT homolog and no homolog of protein kinase C or any other eukaryotic-type protein kinase14
The second branch of the HIT superfamily (), is named for the human FHIT protein which is encoded at the chromosome 3 fragile
. The FHIT branch consists of two enzymes with demonstrated diadenosine polyphosphate hydrolase activity16, 17
and one protein we predict to have such activity. Diadenosine polyphosphate hydrolases cleave dinucleotide polyphosphates such as A(5′)ppp(5′)A that have adenosine or guanosine nucleosides esterified to a chain of 3 to 5 phosphates18
. The phosphate ester linkages are to the 5′-ribose oxygen on both nucleosides such that cleavage of ApppA yields AMP plus ADP, cleavage of AppppG yields AMP plus GTP, and so forth. Phylogenetically, these enzymes constitute a distinct branch of the HIT superfamily that appears to have emerged within eukarya. Proteins within this branch contain 18 conserved residues that differ from those in mammalian HINT. The hydrolases also share a C-terminal extension of 37 to 95 amino acids containing eight invariant amino acids.
The human FHIT
gene at 3p14.2 is disrupted in many human tumors15, 19–28
. As such, FHIT
is a candidate tumor suppressor gene whose loss of function may contribute to the initiation or progression of neoplasias. When FHIT was identified as a member of the HIT protein superfamily15
and subsequently shown to cleave diadenosine polyphosphates17
, the molecular basis of its intrinsic nucleotide binding and hydrolase activity were not understood. The existence, demonstrated herein, of two identical purine mononucleotide-binding sites in a HINT dimer suggests the mode of binding of a dinucleotide by FHIT. Sequence alignments and modeling of FHIT from HINT-nucleotide coordinates have suggested the identities of amino acids in FHIT that could be involved in cleavage of diadenosine polyphosphates.
We report here the crystal structures of HINT in its unliganded form, as well as HINT bound to adenosine, GMP and 8-Br-AMP. These structures show that the residues most conserved in the HIT superfamily are involved in binding nucleotides, and that the histidine triad, rather than constituting a zinc binding site2, 5
, forms part of the phosphate-binding loop. Because HINT has no PKC inhibitory activity (J.G., N. Liang & J.M.L., in prep.), has bacterial11, 14
homologs in organisms without protein kinase C, and has been conserved throughout evolution as a nucleotide-binding protein, we argue that classification of HINT as PKCI-1 is unjustified. We propose that this protein and its branch of the HIT superfamily be called HINT.
We also demonstrate that the dimeric structure of HINT, dominated by a 10-stranded anti-parallel β-sheet, can be superimposed upon the monomeric core structure of galactose-1-phosphate uridylyltransferase (GalT) from E. coli29
, a highly conserved enzyme whose deficiency in humans causes galactosemia30
. Despite a lack of overall sequence similarity, nucleotide binding by GalT is conferred by a subset of the most highly conserved residues in the HIT superfamily. The GalT monomer can be seen as consisting of two repeated HINT domains related by an internal pseudo-two-fold axis. Our analysis suggests that a HINT protein was a common ancestor of FHIT, GalT and other nucleotide-binding proteins. As HINT forms the scaffold for diverse enzymatic activities, it also creates a substructure for dissecting their function.