|Home | About | Journals | Submit | Contact Us | Français|
Escherichia coli diacylglycerol kinase (DAGK) represents a family of integral membrane enzymes that is unrelated to all other phosphotransferases. We have determined the three-dimensional structure of the DAGK homotrimer using solution NMR. The third transmembrane helix from each subunit is domain-swapped with the first and second transmembrane segments from an adjacent subunit. Each of DAGK’s three active sites resembles a portico. The cornice of the portico appears to be the determinant of DAGK’s lipid substrate specificity and overhangs the site of phosphoryl transfer near the water-membrane interface. Mutations to cysteine that caused severe misfolding were located in or near the active site, indicating a high degree of overlap between sites responsible for folding and for catalysis.
E. coli DAGK is encoded by the dgkA gene and catalyzes direct phosphorylation of diacylglycerol (DAG) by MgATP to form phosphatidic acid as part of the membrane-derived oligosaccharide (MDO) cycle(1–3). In Gram-positive organisms, the dgkA homolog encodes an undecaprenol kinase, indicating a role in oligosaccharide assembly or in related signaling pathways(4). The DAGK homolog in Streptoccus mutans is known to be a virulence factor for smooth surface dental caries(5). DAGK was among the first integral membrane enzymes to be solubilized, purified, and mechanistically characterized(6). The wild type protein is very stable(7, 8) and can spontaneously insert into lipid bilayers to adopt its functional fold(9, 10). Paradoxically, DAGK resembles many disease-linked human membrane proteins because it is highly susceptible to mutation-induced misfolding(10–12). DAGK functions as a 40 kDa homotrimer, with a total of nine transmembrane (TM) helices and three active sites(13).
The structure of DAGK was determined using solution NMR methods(14) under conditions in which the enzyme is a functional homotrimer solubilized in ca. 100 kDa dodecylphosphocholine micelles, work that extends other solution NMR studies of >20kDa multi-span membrane proteins (15–22). The backbone structure of the helical TM domain of DAGK (residues 26–121) was precisely determined by the data (Fig. 1, fig. S1 and table S4); however, motions associated with the N-terminus (residues 1–25) have hindered determination of its conformation beyond confirming the presence of two stable amphipathic helices.
DAGK’s structure bears no resemblance to that of the water soluble DAGK(23) (Fig. 1). The three-fold symmetry axis lies at the center of a parallel left-handed bundle formed by the second transmembrane (TM2) helices of the three subunits. TM2 has previously been proposed to play a central role in DAGK’s folding and stability(24, 25) and contains several highly conserved residues (Fig. 1), particularly near the membrane/cytoplasm interface. Characterization of a series of cysteine-replacement mutants for sites in TM2 showed that mutations in this segment often resulted in a dramatic reduction in catalytic function (Fig. 2 and table S1), underscoring the importance of TM2 to DAGK folding and catalysis.
DAGK is seen to be a domain-swapped homotrimer (Fig. 1C). TM3 makes virtually no contact with TM2 from the same subunit. Instead, it packs against the hairpin formed by the first and second TM segments from an adjacent subunit (TM1′ and TM2′). Domain-swapping may contribute to the high stability of wild type DAGK(7, 8). Conversely, domain-swapping may illuminate why DAGK mutants are often difficult to refold following denaturation(11).
A distinctive structural feature of the DAGK structure is a membrane-submerged cavity that resembles a portico (Figs. 1A and and2B2B and fig. S1). TM1 and TM3 serve as the pillars that bound the entry, terminating at an inner wall formed by TM2′ from a neighboring subunit, and crowned by an overhanging cornice comprised of the connecting loop between TM2 and TM3. The portico, and in particular the cornice, contains a majority of the residues seen to be functionally essential in our mutagenesis studies (Fig. 2). Each portico contains functionally-critical residues that are contributed by two different subunits, consistent with a previous mutagenesis study which showed that each active site is shared between subunits (13). Titrations of DAGK with its substrates/products (MgATP, DAG, and phosphatidic acid) and with a non-hydrolyzable ATP analog (β, γ-methyleneadenosine 5′-triphosphate, AMP-PCP) were monitored by NMR. Significant and saturable changes in NMR resonances upon ligand binding (Fig. 3 and fig. S5) confirmed that the portico includes the lipid substrate binding site of DAGK. The overhanging cornice likely blocks lateral diffusion into the active site of lipids with head groups larger than DAG and phosphatidic acid. On the other hand, the spaciousness of the lower part of the portico explains the lack of specificity of DAGK with respect to the acyl chain composition of diglyceride substrates (26). Interactions of membrane enzymes with membranous substrates more typically involve gated internal cavities within the protein or well-structured grooves that confer high specificity for specific substrates (27–29), although the active site of the homodimeric beta-barrel outer membrane phospholipase A also exhibits a portico-like architecture, consistent with the lack of specificity of this enzyme in terms of substrate acyl chains(27,30).
NMR resonances for three residues located just under the cornice on TM1 and TM2′ exhibit significant shifts in response to the binding of nucleotides and lipids (Fig. 3). These sites are likely proximal to the site of ATP-to-DAG phosphoryl transfer, consistent with the observed proximity to the cornice of many of the highly conserved and functionally-critical residues. We suggest that a second role for the cornice in the active site may be to seal off the substrate-filled active site from water (to avoid ATP hydrolysis), a catalytic imperative that is usually satisfied in water soluble phosphotransferases, such as adenylate kinase, by global domain motions that close the active site upon substrate binding(31). DAGK appears not to undergo a global conformational change, as revealed by observation of only modest and highly localized changes in NMR resonance positions for DAGK upon forming binary complexes with its substrates or with a ternary complex with DAG and MgAMP-PCP (Fig. 3 and fig. S5).
As alluded to above, we systematically examined the functional consequences of mutating each residue in DAGK to cysteine to gain insight into structure-function relationships beyond what can be deduced either from multiple sequence alignment or from a previous phenotype-based random mutagenesis study(32). Mutants that resulted in >85% loss of catalytic activity (listed in table S1) were further characterized to reveal two categories of defects (details in table S2 and summary in Fig. 2). First are mutations that do not disrupt protein folding, but result in loss of catalytic function. These residues are either directly in the active site or play specific local structural roles in support of the active site. Second are mutations to cysteine that result in misfolding so severe that in vivo expression levels were seen to be reduced (Fig. 2 and table S2), often to an undetectable level. In cases where purification was possible, such mutants were typically observed to be aggregation-prone (table S2). While cysteine-scanning mutagenesis is a commonly used in studies of membrane proteins (c.f. 33), we are unaware of previous documentation of a set of mutants analogous to the misfolding-prone variants described in this work.
A remarkable feature of the DAGK structure is the close proximity between the active site and a majority of residues for which mutations to cysteine result in severe misfolding (Fig. 2 and table S2). This is in contrast with enzymes, such as those of the TIM barrel superfamily(34), where the active site is set within a robust structural framework that is tolerant of major remodeling of catalytic residues. Given that numerous diseases, such as cystic fibrosis, are associated with inherited or sporadic mutations that result in single amino acid changes in membrane protein sequence(12), our results serve as a warning that even for a structurally well-characterized membrane protein, knowledge regarding the location of a disease-promoting mutation site cannot a priori be used to reliably predict the exact nature of the molecular defect that is linked to disease etiology (i.e., local active site disruption vs. catastrophic misfolding).
That most of the residues important for avoiding misfolding of DAGK are located at the DAGK active site suggests that the portico is unlikely to be adapted by evolution to orthologous functions. While DAGK has been shown to be a highly efficient catalyst in its microbial physiological niche(3), intimate linkage between activity and folding may help to explain why this enzyme is an evolutionary orphan.