Tuesday Oct 9 9 pm Eastern
Wednesday Oct 10 8 pm BST, 12 noon Pacific
Science. 2018 Sep 7;361(6406). pii: eaat9819. doi: 10.1126/science.aat9819. Epub 2018 Aug 9.
Structure of the human PKD1-PKD2 complex.
Autosomal dominant polycystic kidney disease (ADPKD) is the most commonly inherited kidney disease. It is defined by the replacement of normal kidney tissue with cysts, leading eventually to end-stage kidney disease (ESKD). Here are some key statistics:
ADPKD is the 4th leading cause of ESKD in the USA
In Europe, 1 in 10 patients with ESKD have ADPKD
In the USA, Australia, and New Zealand 1 in 20 patients with ESKD have ADPKD
ADPKD is caused by defects in the proteins polycystin 1 (PC1, also referred to as PKD1) or polycystin 2 (PC2, also referred to as PKD2) that form the polycystin complex encoded by the PKD1 and PKD2 genes, respectively. Mutations in PKD1 and PKD2 account for 85% and 10% of all cases of ADPKD. Around 15% of patients with ADPKD do not have a family history of cystic disease and carry a de novo PKD1 or PKD2 mutation. Despite the importance of these genes and their resulting proteins, how their structures interfaced and interactd was unknown until Su et al.’s recent publication in Science. This landmark paper is the subject of the October 9 and 10 #NephJC chats.
Defective PKD1 or PKD2 result in abnormal primary ciliary function in tubular epithelial cells, involving aberrant cell signaling and increased cellular proliferation, with the result being the formation and growth of renal cysts. Primary cilia contain a microtubule skeleton that acts as a mechanical and chemical sensor (Primary cilia was briefly reviewed in the Ciliopathies Scouting Report in NephMadness 2017). Tubular flow across the primary cilia causes them to bend, which increases intracellular calcium levels.
How does this happen? PKD1 is an integral membrane receptor, and PKD2 is an integral transmembrane protein located mostly in the endoplasmic reticulum (ER), which transports calcium. PKD1 and PKD2 have been found to colocalize, supporting the idea that they form an ion channel complex. One prevailing hypothesis on pathophysiology is as follows: if the polycystin complex is disturbed and calcium influx into the cell is impaired, there is subsequent increase in cAMP levels, mTOR activation and an increase in cyst formation.
As ADPKD cysts grow they compress normal kidney parenchyma, causing nephron loss and fibrosis. ADPKD can be screened for using clinical imaging of the kidneys in patients with a family history of ADPKD over the age of 40. Genetic testing is also available. As ADPKD progresses, the patient develops urinary concentrating defects, polyuria, nocturia, nephrolithiasis (kidney stones), hypertension, infections, and progressive loss of kidney function.
Various medications are used or being studied to slow cyst development in ADPKD. These include V2 receptor antagonists, statins, somatostatin analogues (e.g. octreotide), mTOR inhibitors, antihypertensives, and others. Management of ADPKD also involves controlling secondary effects of the disease. Dietary interventions such as increasing water intake and salt restriction may also slow the rate of cyst growth.
However, these therapeutic strategies do not halt or reverse ADPKD as they do not target the direct cause of disease – DNA variants in PKD1 and PKD2. To understand fully how a protein such as PKD1 functions and interacts, one must understand the protein's structure as well as the structure of the complex it forms with other key proteins. This biochemical knowledge is essential for the subsequent development of targeted therapeutics. Additionally, the exact mechanistic underpinnings of how ADPKD variants alter PKD1 and PKD2 function has remained elusive. Now, in combination with the recently solved PKD2 structure, Su et al.’s solving the crystal structure of the complex will allow mapping of causal DNA variants onto this 3D structure. This will not only facilitate our understanding of ADPKD on a molecular level, but offers a promising platform for developing therapeutic strategies tailored to individual variants, raising the prospect of personalized medicine.
The authors of this paper used cryo-electron microscopy (cryo-EM) to solve the structure of the PKD1/PKD2 complex. Cryo-EM allows scientists to visualize protein structures that was not possible using X-ray crystallography and Nuclear Magnetic Resonance (NMR) spectroscopy. X-ray crystallography cannot be used for some proteins that don't crystallize and for other proteins that undergo structural alterations in the crystallization process that render the diffraction process inaccurate. NMR spectroscopy is limited to smaller proteins, so is not applicable to the larger PKD1 and PKD2 proteins. Cryo-EM has revolutionized structural biology to the point that the scientists contributing to the development of cryo-EM won the 2017 Nobel Prize in Chemistry.
How does cryo-EM work? In short, transmission electron microscopy (TEM) is used to gather structural information on the atomic level of proteins that have been frozen. Proteins must be frozen for this process because their structural details cannot withstand the harsh electron beams from TEM. However, traditional freezing would not work because crystalline ice would diffract an electron beam obscuring structural information. The protein needs to be flash-frozen in liquid ethane, which causes the protein samples to freeze so rapidly that the surrounding water forms disordered glass that does not diffract the microscope's beams.
Prior to cryo-EM, an abundant amount of the desired protein needs to be purified. Because a large amount of protein is required for visualization, the PKD1/PKD2 complex, which is lower in abundance than other proteins, was not extracted from human tissue or animal models. Instead, it was overexpressed and purified from a large amount of cells cultured in the laboratory (40-50 liters of cell culture for the PKD1/PKD2 complex). In addition, the flexible regions of the protein were not included so that protein solubility was optimized, leaving only the interacting portions of the proteins to be visualized.
As Dr. Paul Welling eloquently notes in his recent perspective piece, a popular hypothesis for the PKD1/PKD2 complex has been that PKD2 proteins form a cation pore while PKD1 regulates the channel. This is not an unexpected hypothesis given PKD2's structural similarities to transient receptor potential (TRP) channel proteins that have been studied in the context of calcium signaling in podocytes.
Because the intricate details of structural biology are beyond the scope of the upcoming #NephJC discussion, we will summarize the main highlights of the results here. For more details, please watch the summary video linked here.
The truncated PKD1/PKD2 complex consists of one PKD1 and 3 PKD2 molecules, consistent with prior lower resolution characterization of the full-length proteins in complex.
A Novel Segment in PKD1 May Affect Function of PKD1/PKD2 Complex
On the left, the purple arrows point to structural differences between the S6 segment of PKD2 and the S6a and S6b segments of PKD1; the angles and folding of the helix differ. In addition, the S6a and S6b segments of PKD1 house positively charged residues (red circles on right) that face the cavity of the complex, which would disfavor cation (such as calcium) penetration. The S6 segment of PKD1 does not resemble other channel structures, calling into question the popular hypothesis that the PKD1/PKD2 complex would allow calcium flux.
PKD1 Is Missing TLC Element that Is Needed to Interface with PKD2
Because PKD1 is missing a protein feature (TLC) that is needed to interface with PKD2 on both sides, there is a gap between one side of PKD1 and PKD2 TOP domains. Because of this gap, PKD1 likely could not assemble with PKD2 in a symmetric 2:2 ratio as that would introduce another gap in the complex and be biochemically unfavorable.
Mapping ADPKD Variants onto the Solved PKD1/PKD2 Complex
As stated above, most of the disease-causing variants are in the PKD1 gene. Surprisingly, none mapped to the pore-forming S6 portion of PKD1. All of the colored spheres represent documented ADPKD variants.
FOOD FOR THOUGHT
Now that we have seen what structural biology has elucidated in terms of PKD1/PKD2 complex structure, the functional work for what this all means needs to commence. Although we have more structural information, this paper did not present mechanistic data in terms of cell physiology and direct contributions to ADPKD. How does the 1:3 stoichiometry play into cellular function? Is the complex not an ion channel? How do ADPKD-associated DNA variants affect the protein and potentially structure-function? These questions and more will be discussed at the #NephJC chat.