polyphosphates

Inorganic polyphosphate – an ubiquitous but enigmatic phosphate polymer

Figiure 1. Schematic view of an inorganic polyphosphate polymer.

Figure 1. Schematic view of an inorganic polyphosphate polymer.

Pro- and eukaryotic organisms rely on phosphate as a major building block of their DNA, lipids, nucleotides and proteins and thus require to sense, uptake, metabolize and store inorganic phosphate. One rather poorly understood storage form of inorganic phosphate in all pro- and eukaryotic cells is inorganic polyphosphate, a linear chain of orthophosphate units linked by phosphoanhydride bonds (Figure 1). In bacteria, polyP is generated from ATP by polyP kinase  (Sylvie Kornberg, BBA, 1957) and serves important physiological roles (Kornberg et al., Ann Rev Biochem, 1999) . PolyP kinases however are absent from eukaryotic genomes, and thus it was unknown for a long time how eukaryotes would synthesize inorganic polyphosphates.

A vacuolar integral membrane protein complex synthesizes polyP in yeast

Figure 2. Molecular architecture of the VTC subunit Vtc4. An N-terminal SPX domain is followed by the catalytic polyphosphate polymerase domain and a transmembrane domain, which allows for the transport of the growing polymer into the vacuolar lumen.

Figure 2. Molecular architecture of the VTC subunit Vtc4. An N-terminal SPX domain is followed by the catalytic polyphosphate polymerase domain and a transmembrane domain, which allows for the transport of the growing polymer into the vacuolar lumen.

Serendipity lead us to discover the first eukaryotic enzyme that would synthesize inorganic polyphosphate (Hothorn et al., Science, 2009). It turned out to be an integral membrane protein complex embedded in the vacuolar membrane, not a soluble enzyme. The so-called Vacuolar Transporter Chaperone (VTC) Complex (a nice name for “we don’t know what this protein is doing”) consists of at least three different subunits. All subunits have a transmembrane domain (see below). Two subunits (Vtc2/3 and 4) in addition have soluble portions, which face the yeast cytoplasm (Mueller et al., EMBO J, 2002) (Fig. 2). Using X-ray protein crystallography, we could demonstrate that the soluble portions in VTC contain a tunnel-shaped triphosphate tunnel metalloenzyme (TTM) domain. We found that the Vtc4 TTM domain would bind and hydrolyze ATP in the presence of a manganese metal cofactor. After dialysing the tunnel domain in a solution containing ATP, we obtained crystals of Vtc4. To our surprise these crystals did not contain ATP bound in the catalytic cleft, but a long phosphate polymer (Fig. 3) (Hothorn et al., Science, 2009).

Figure 3. Ribbon diagram of the Vtc4 catalytic polymerase domain. A long phosphate polymer is bound in the active site cleft.

Figure 3. Ribbon diagram of the Vtc4 catalytic polymerase domain. A long phosphate polymer is bound in the active site cleft.

VTC turned to be the long sought-for polyphosphate polymerase in yeast. Yeast cells contain high concentrations of polyphosphate in their vacuole. Point-mutations in the VTC transmembrane domain inhibit translocation of the polymer into this storage compartment. Upon inactivation of polyphosphate polymerisation in VTC, yeast mutants cannot survive under phosphate limiting conditions and have multiple cellular defects. We can thus speculate that polyphosphates are important storage forms of phosphate in eukaryotic cells. They may also play roles in ion homeostasis and energy metabolism.

The metabolism and cellular functions of polyphosphates in higher eukaryotes

Unfortunately, the VTC complex is only conserved in unicellular eukaryotes and not in plants or animals. We are thus now trying to find polyphosphate metabolizing enzymes in higher organisms, namely in our model plant Arabidopsis thaliana. We are also interested to find polyphosphates in plants and understand their cellular and physiological functions (supported by an ERC starting grant).

Arabidopsis contains AtTTM3, a soluble enzyme with structural similarity to yeast Vtc4.

Figue 4. The catalytic center of AtTTM3 contains two metal binding sites, one of which is involved in substrate coordination. The second metal activates a water molecule required for the hydrolysis reaction.

Figure 4. The catalytic center of AtTTM3 contains two metal ions, one of which is involved in substrate coordination. The second metal activates a water molecule required for the hydrolysis reaction.

We used a structure-guided approach to find proteins in Arabidopsis with similarity to Vtc4. There are three TTM proteins present in the Arabidopsis genome (AtTTM1-3), with TTM3 being a small soluble enzyme with high structural similarity to Vtc4 (Martinez, Truffault & Hothorn, JBC, 2015). Postdoctoral fellow Jacobo Martinez found however, that instead of polymerizing polyPs from ATP, AtTTM3 efficiently hydrolyzes short-chain polyPs in vitro. Why this discrepancy in catalytic activity between the yeast and plant enzymes? Jacobo dissected the catalytic mechanism of plant, yeast, bacterial and human TTM enzymes in mechanistic and structural detail. He found that most TTM enzymes hydrolyze their substrates using a two metal catalytic mechanism (Figure 4).

Figure 5. Schematic view of the tunnel domain in different TTM enzymes, which allow their substrates to enter the tunnel from opposite sites, thus generating different leaving groups.

Figure 5. Schematic view of the tunnel domain in different TTM enzymes, which allow their substrates to enter the tunnel from opposite sites, thus generating different leaving groups.

Vtc4 however, uses only one catalytic metal, with the second metal binding site modified in such a way, that it can harbor a phosphate polymer to which the ATP γ-phosphate can be transferred to (Martinez, Truffault & Hothorn, JBC, 2015). Jacobo’s work also allowed us to understand, how TTM enzymes can carry out such a wide array of enzymatic reactions (adenylate cyclase, thiamine triphosphatase, tripolyphosphatase etc. reactions have been reported): different TTM enzymes let their substrates enter from opposite sides of the tunnel domain. Thus, with the core catalytic center always remaining in the same location, TTM enzymes can generate either phosphate or pyrophosphate leaving groups (Figure 5).

SPX domains are inositol pyrophosphate sensor domains controlling phosphate homeostasis in eukaryotic cells

Rebekka Wild, a PhD student in the lab, finally figured out what the N-terminal SPX domains in VTC and other eukaryotic proteins actually do (Wild et al., Science, 2016).  SPX domains are small, soluble domains found in fungi, plants and animals. They can exist as stand-alone modules but are often located at the N-termini of proteins involved in phosphate uptake, transport, storage, metabolism or signaling. We determined structures of fungal and human SPX domains from crystals obtained by carrier driven crystallization (Wild & Hothorn, Protein Science, 2016). The different structures revealed a new fold with two long a-helices, connected by linkers of variable size. These core helices and two smaller C-terminal helices together form a 3-helix bundle, which is preceded by an flexible N-terminal helical hairpin (Figure 6). Many invariant lysine residues, which represent sequence fingerprints for SPX domains, are clustered in proximity to the N-terminal hairpin structure. A combination of genetic and biochemical experiments in yeast and Arabidopsis revealed that this basic surface represents a docking platform for inositol pyrohosphosphates (PP-InsPs), signaling molecules with poorly characterized cellular functions (Figure 6).

The concentrations of PP-InsP are known to change in cells, depending on whether they are supplied with sufficient amounts of inorganic phosphate or whether they experience phosphate starvation. We could demonstrate that SPX domains, when bound to PP-InsPs can interact with other proteins. In plants, one such interaction partner is a transcription factor, which is switched on under phosphate starvation to induce expression of genes that allow the plant to respond to the lack of this important nutrient. Under normal growth conditions, when PP-InsP levels are high, the transcription factor is kept inactive by forming a PP-InsP-dependent complex with a plant SPX domain. Under phosphate starvation, the lack of PP-InsP leads to dissociation of the transcription factor – SPX domain complex, thereby enabling the transcription factor to transcribe its target genes. Taken together, our work defines SPX domains as cellular receptor for PP-InsPs, which control phosphate uptake, transport, storage, metabolism and signaling in fungi, plants and animals.

Figure 6. (left panel) Crystal structure of the SPX domain of C. thermophilum glycerophosphodiesterase SPX domain (ribbon diagram) in complex with inositol hexakisphosphate (in bonds representations). (right panel) Close-up view of the PP-InsP binding site, with many conserved lysine residues (in bonds representation) coordinating the phosphate groups of the ligand (dotted lines).