Heme-binding b5-domain proteins are found in all major eukaryotic lineages. A bacterial cytochrome b5
homolog, cytochrome b558
from Ectothiorhodospira vacuolata,
has also been identified and structurally characterized [20
]. The heme-binding b5-domains are involved in electron transfer to and from other proteins, or other domains on the same protein (as in flavocytochrome b2
] and sulfite oxidase [22
]). The heme ligand is buried in the binding pocket formed by the two pairs of α helices over the β sheet, and is coordinated by the NE atoms of two conserved histidine residues [21
]. The positions of these two histidines are indicated in Figure .
Neither of the MAPR, HERC2, or chitin synthase b5-domains appear to bind heme or to be involved in redox reactions. Indeed, they all lack the pair of heme-coordinating histidine residues. Chitin synthases catalyze the polymerization of N-acetylglucosamine through β-1,4 linkage. Chitin is an important constituent of fungal cell walls. Chitin synthase genes are present in various numbers in different fungal species, and the relative importance of the individual isozymes in particular fungal species is only partially understood. We have identified several fungal chitin synthases with a b5-domain, which can be organized into three groups on the basis of domain organization (Figure ).
The function of the giant HERC2 protein is not known. It has been predicted to act as a guanine-nucleotide-exchange factor and an E3 ubiquitin ligase, involved in intracellular protein trafficking and degradation [23
As mentioned above, heme-binding b5-domains are found in all eukaryotic lineages. The MAPR proteins appear to be restricted to plants and metazoans, and the HERC2 proteins with a b5-domain are mammalian. The b5-domain chitin synthases are only found in fungi. Taken together, our findings suggest that the heme-binding cytochrome b5 domain may have served as a template for the more recent evolution of novel ligand-binding pockets, such as a steroid-binding site in the MAPR proteins. We predict that the b5-domains that we have identified in chitin synthases and HERC2 proteins might serve as binding sites for lipid ligands. The relatively narrow species distributions of b5-domain chitin synthases and HERC2 proteins suggests that these binding sites may have arisen as a result of relatively recent insertions of a b5-domain. We did not find a b5-domain in the HERC2-related proteins HERC1 or HERC3.
Most of the proteins with a b5-domain are linked to cell membranes, either directly or by forming part of membrane-associated complexes. We propose that the proximity of cell membranes to a heme-binding template pocket could have served to give rise to new ligand-binding pockets with specificity for membrane-soluble molecules such as steroids. Thus, MAPRs may represent an adaptation by which cells could start making use of steroids as triggers for rapid response mechanisms.
The MAPRs could be localized to both plasma and intracellular membranes [11
], which would afford cells greater flexibility in utilizing progesterone and other ligands for rapid signaling. It seems likely that MAPRs bind to progesterone intracellularly: all the other b5-domains are intracellular, and there is no evidence to suggest that the MAPR b5-domain is extracellular. It is also probable that another class of membrane-associated progesterone receptors can bind to their ligand extracellularly: the rapid effects of progesterone on spermatozoa may be mediated by such a receptor, and are reproducible even with preparations of progesterone to which the plasma membrane is impermeable (reviewed in [24
]). Antibodies raised against the carboxy-terminal ligand-binding domain of the nuclear progesterone receptor identified a surface protein in human spermatozoa, and were able to inhibit the rapid effects of progesterone [25
]. Antibodies raised against the amino-terminal transactivation domain of the nuclear progesterone receptor did not identify any such protein in human spermatozoa [26
]. These findings suggest that spermatozoa have a cell-surface progesterone receptor with a classical ligand-binding domain, but without a transactivation domain. Unfortunately, no sequence data for this protein are available, and it is not known whether a similar protein is expressed in other tissues.
The details of the physiological role of the MAPRs remain to be worked out. For example, mutagenesis studies should help better to define the importance of individual residues in the MAPR molecule. The determination of the molecular structure of MAPR would provide crucial information on the nature of the interaction of MAPR and its ligand, as well as its possible interaction with the intracellular signaling machinery. Sequencing and structural determination of the extracellular sperm progesterone receptor would allow comparison between it, MAPR, and the nuclear progesterone receptor. Supplementing these data with detailed studies of the tissue-expression patterns of these molecules, and their associated signal transduction mechanisms should help to provide a better understanding of rapid cellular signaling by steroids.