Macromolecular assembly of polycystin-2 intracytosolic C-terminal domain Frederico M. Ferreiraa,b,1, Leandro C. Oliveirac, Gregory G. Germinod, José N. Onuchicc,1, and Luiz F. Onuchica,1 aDivision of Nephrology, University of São Paulo School of Medicine, 01246-903, São Paulo, Brazil; bLaboratory of Immunology, Heart Institute, University of São Paulo School of Medicine, 05403-900, São Paulo, Brazil; cCenter for Theoretical Biological Physics, University of California at San Diego, La Jolla, CA 92093; dNational Institute of Diabetes, Digestive, and Kidney Diseases, Bethesda, MD 20892-2560 Contributed by José N. Onuchic, April 28, 2011 (sent for review March 20, 2011) Mutations in PKD2 are responsible for approximately 15% of the In spite of the aforementioned information and insights, the autosomal dominant polycystic kidney disease cases. This gene macromolecular assembly of PC2t homooligomer continued to encodes polycystin-2, a calcium-permeable cation channel whose be an open question. In the current work, we present the most C-terminal intracytosolic tail (PC2t) plays an important role in its comprehensive set of analyses yet performed and that show interaction with a number of different proteins. In the present PC2t forms a homotetrameric oligomer. We have proposed a study, we have comprehensively evaluated the macromolecular PC2 C-terminal domain delimitation and submitted it to a range assembly of PC2t homooligomer using a series of biophysical of biochemical and biophysical evaluations, including chemical and biochemical analyses. Our studies, based on a new delimitation cross-linking, dynamic light scattering (DLS), circular dichroism of PC2t, have revealed that it is capable of assembling as a homo- (CD) and small angle X-ray scattering (SAXS) analyses. We have tetramer independently of any other portion of the molecule. also used molecular dynamics simulations employing an all-atom Our data support this tetrameric arrangement in the presence and structure-based model (SBM) to evaluate the macromolecular as- absence of calcium. Molecular dynamics simulations performed with sembly of PC2t, by comparing the structures obtained from the a modified all-atoms structure-based model supported the PC2t tet- simulation including the raw SAXS data and by reconstructing rameric assembly, as well as how different populations are disposed the distribution of states in solution using a system forced by en- in solution. The simulations demonstrated, indeed, that the best- tropy. The simulations determined, in fact, that the best-scored BIOPHYSICS AND scored structures are the ones compatible with a fourfold oligomeric structures are also the ones compatible with tetramerization. state. These findings clarify the structural properties of PC2t domain COMPUTATIONAL BIOLOGY and strongly support a homotetramer assembly of PC2. Results In Silico Analysis of PC2t Molecular Properties. The domain topology utosomal dominant polycystic kidney disease (ADPKD) is of PC2 was initially examined upon the analysis of its primary Athe most common monogenic life-threatening human dis- sequence (Fig. 1). In silico analysis of PC2t polypeptide sequence ease, with a prevalence of 1∶400 to 1∶1;000 (1). Whereas muta- revealed an inverse correlation between hydrophobicity and tions in the PKD1 gene are responsible for approximately 85% tendency of disorder, with significantly low hydrophobicity and of the disease cases, nearly 15% are caused by pathogenic muta- high tendency of disorder in the transition between the trans- tions in PKD2 (1). The PKD2 gene encodes polycystin-2 (PC2), a membrane and intracellular domains, aa 649–681 (Fig. S1). A 968-amino acid (aa) membrane protein with six transmembrane similar pattern was observed for flexibility, particularly for the helices (TM) and both C- (PC2t) and N-termini intracytosolic interface residues between these two domains, where the residues (1, 2). PC2 and the membrane protein polycystin-1 (PC1), the are highly hydrophobic and structurally organized (Fig. S2). The PKD1 gene product, are thought to physically interact by their PC2t middle region (aa 800–830) shows remarkably high average C-termini (3). In recent years, a series of studies have implicated flexibility and tendency of disorder, where a linker is positioned the primary apical cilia in the pathogenesis of polycystic kidney between the EF-hand and the coiled-coil subdomains. The same disease. PC2 is a member of the transient receptor potential behavior was detected in the PC2t final region (aa 940–968). (TRP) channel superfamily (TRPP2), acting as a nonselective ca- These results, therefore, support the assignment of the intracyto- tion channel involved in calcium transport (1, 4, 5). The PC1–PC2 plasmic PC2t domain to the 289 C-terminal residues 680–968. complex plays a key role in this ciliary mechanism, by apparently It must be noted that PC2t is highly charged, with a number of translating physical or chemical stimuli in calcium influx through negatively charged residues yielding theoretical pI of 5.1 and PC2, a process that is followed by significant calcium release from molecular mass of 32.6 kDa. intracellular stores (6). The PC2 topology predicts the pore structure to reside in TMs Dynamic Light Scattering. To access the monodispersity of PC2t 5 and 6, whereas a sensor portion is expected in TMs 1–4 (2, 7). samples and for a rough estimation of its maximum dimension, TRP channels have been shown to form homo and/or heteromul- we performed DLS experiments. Such experiments revealed the timers, a property apparently correlated with quantitative and PC2t samples in the monodispersed state. The estimation of the qualitative aspects of their function (8). Full-length PC2 appears oligomer maximum dimension was 17 Æ 4 nm. to follow this pattern, because a recent study supports PC2 assem- bling as a homotetramer and as a PC2-TRPC1 heterotetramer PC2t Expression, Purification, and Molecular Mass Estimation. The (7). Previous work has suggested that a coiled-coil subdomain coding sequence corresponding to PC2t was amplified by identified in the PC2t (PC2t-CC) plays critical roles in PC2 oli- gomerization and in its physical interaction with PC1 to form the PC1–PC2 complex, a process that also involves a coiled-coil Author contributions: F.M.F., L.C.O., G.G.G., J.N.O., and L.F.O. designed research; F.M.F. and L.C.O. performed research; F.M.F., L.C.O., J.N.O., and L.F.O. analyzed data; and subdomain in PC1t (1). Another recent study, however, suggests F.M.F., L.C.O., G.G.G., J.N.O., and L.F.O. wrote the paper. þþ that the PC2t-CC assembles as a homotrimer (9). A Ca -bind- The authors declare no conflict of interest. ing EF-hand subdomain has also been predicted in PC2t and 1 þþ To whom correspondence may be addressed. E-mail: [email protected], [email protected], been proposed to function as a Ca -sensing switch (2, 10), or [email protected]. whereas the PC2 channel activity has been shown to be regulated This article contains supporting information online at www.pnas.org/lookup/suppl/ by calcium (4, 5, 11). doi:10.1073/pnas.1106766108/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1106766108 PNAS ∣ June 14, 2011 ∣ vol. 108 ∣ no. 24 ∣ 9833–9838 Downloaded by guest on September 25, 2021 to chemical cross-linking assays, shown in Fig. 2D. A blurred band corresponding to the PC2t oligomer was detected, with a middle- position height slightly above the 116-kDa marker. The profile of the log(MM) vs. Rf plot is sigmoidal, so the assumption of line- arity is valid only for a finite but useful range (Fig. S5). Because the nonlinearity is even more pronounced for the higher MM markers, a polynomial regression was applied. The apparent mass Fig. 1. (A) Topology of the PC2 predicted domains: in green, the cytosolic calculated for the cross-linked sample was 123 kDa, a mass com- domains; in cyan, the putative extracellular domain; and in magenta, the patible with a tetramer, whereas a value of 36 kDa was obtained transmembrane domain. (B) Topology of PC2t subdomains and motifs as for the negative control sample in accordance to that expected for predicted by PRODOM: in red, EF-hand subdomain and its motif (arrow); a monomer. and in yellow, the coiled-coil subdomain and its motif and the MS sequenced residues (arrow). Structural Features of PC2t Monomer Revealed by Mass Spectroscopy. C standard PCR and cloned in an expression vector. Successful Fig. 2 shows the PC2t controlled proteolysis with trypsin, reveal- PC2t overexpression followed by western blot analysis of the so- ing a structural core of approximately 14.4 kDa insensitive to such luble fraction of cell lysate are shown in Fig. 2A, confirming a a proteolytic effect. This finding is, in fact, inconsistent with the in band immediately above the 30-kDa marker. silico trypsinization pattern obtained for PC2t primary sequence, The PC2t molecular mass (MM) estimation from size-exclu- which predicts 40 restriction sites leading to peptides with masses sion chromatography (SEC) revealed an apparent mass of 354 ranging from 147.1 Da (a lysine) to 2845.4 Da (27 residues). The kDa, a mass consistent with an oligomer of order 11 (Fig. S3). observed data require residues organized in a tridimensional The PC2t MM estimated by native gel electrophoresis (N-PAGE; molecular folding, making the remaining arginine and lysine re- Fig. 2B), on the other hand, was 137 kDa, a value consistent with striction sites not accessible to trypsin activity. Interestingly, the a tetrameric arrangement (Fig. S4). The presence of calcium presence of this molecular core was not originally expected, given the intrinsically disorder probability of the primary sequence. neither significantly altered PC2t retention time obtained in SEC Mass spectroscopy analysis determined the sequence of two experiments nor in the migration pattern observed in N-PAGE peptides resulting from the PC2t macromolecular core.