Structural Biology of Cholesterol Homeostasis
Cholesterol is an essential component of our cell membranes. The human liver is capable of producing all the cholesterol we need, but those of us consuming a typical Western diet get about one-third of our cholesterol from food. The liver combines cholesterol and cholesteryl esters into lipoproteins for distribution throughout the body. The predominant form in the bloodstream is the low-density lipoprotein (LDL), which in addition to cholesterol and cholesteryl esters, contains phospholipids, triglycerides, and one copy of apolipoprotein B.
LDL enters cells by receptor-mediated endocytosis, a process in which LDL receptors bind LDL at the cell surface and are enclosed in intracellular vesicles, called endosomes. Once the endosomes have formed, proton pumps generate acidic conditions in their interior (the pH drops from 7.5 to less than 6), which lead to the release of LDL from the receptor. The empty receptor molecules return to the cell surface, while LDL is decomposed and processed inside the cell. The LDL receptor is a crucial part in an elaborate regulatory system that balances cholesterol synthesis and uptake, ensuring on the one hand that cells always have the cholesterol they need and on the other hand that LDL does not accumulate in the blood. One of the most frequent genetic diseases known is familial hypercholesterolemia, which is caused by disabling mutations in the gene encoding the LDL receptor protein. One in about 500 people has such a mutation in one of their chromosomes.
The human LDL receptor, a protein of 839 amino acids, consists of a series of structural modules that are connected by short linker regions. Starting from the amino terminus, there are seven ligand-binding modules, called R1 to R7, of 40 amino acids each. Following these is the epidermal growth factor (EGF) precursor homology region (residues 293 to ~695), which consists of EGF-like repeats A and B, a β-propeller module, and the EGF-like repeat C. Next is the O-linked sugar region, a stretch of 58 amino acids rich in threonine and serine to which carbohydrate molecules are attached. The last two modules are a membrane-spanning region and an intracellular domain; the latter is important for internalization of the receptor. Our low-pH crystal structure of the extracellular portion of the LDL receptor showed the protein in an inactive conformation, with the modules R4 and R5 of the ligand-binding region contacting the surface of the β-propeller module. It provided a plausible model for the binding of LDL at neutral pH and its release at low pH.
It was a great surprise when PCSK9, a serine protease with a distinct influence on cholesterol homeostasis, was discovered in 2003. Research in the laboratories of my colleagues Jay Horton and Helen Hobbs (HHMI, University of Texas Southwestern Medical Center at Dallas) showed that PCSK9 forms a complex with the LDL receptor and that mutations in PCSK9 could either shorten or prolong the lifetime of LDL receptors. Particularly striking was the discovery that people who lacked a functional PCSK9 had extremely low blood cholesterol levels, and, despite other risk factors, had virtually no heart disease.
Hyock Kwon, in collaboration with the Horton laboratory, crystallized a complex of PCSK9 with a fragment of the LDL receptor. This structure defines an interface between the two proteins that could serve as a first target for drugs that could interrupt this interaction. The hope would be that this would lead to a prolonged lifetime of LDL receptors and thus more efficient clearance of LDL from the bloodstream, with beneficial effects for cardiovascular health.
Other current projects related to cholesterol homeostasis include proteins that can sense cholesterol concentration in the endoplasmic reticulum membrane, such as the membrane-bound portions of the proteins HMG-CoA reductase and SCAP, which show sequence similarity in five membrane-spanning segments; this and their interaction with the protein INSIG suggest a common mechanism for cholesterol sensing. In addition, Kwon recently determined the structure of the cholesterol-binding N-terminal domain of the lysosomal protein NPC1, which is part of a cholesterol transport system; disabling mutations of NPC1 cause Niemann-Pick disease. These projects are part of a long-term collaboration with Michael Brown and Joseph Goldstein (University of Texas Southwestern Medical Center at Dallas) and their colleagues.
Innate Immunity of Insects
The innate immune response, which relies on the "hardwired" recognition of distinctive features of pathogens, has been recognized as essential for immunity in a wide range of organisms, including insects and humans. Our first projects in this area have been peptidoglycan-recognition proteins (PGRPs) from Drosophila, which bind components of bacterial cell walls and activate signaling pathways that lead to the synthesis of antimicrobial peptides.
Several crystal structures elucidated structural features of PGRPs and their interactions with peptidoglycan components of bacterial cell walls. Of special interest was the structure of a ternary complex of two PGRPs and a tracheal cytotoxin from Bordetella that showed the principles of signaling by these essential constituents of the innate immune system.
PGRPs are part of a detection system for foreign intruders; their signals cause the expression of sets of proteins and peptides that together can kill pathogens. One class of proteins in these sets comprises the thioester-containing proteins, which when activated can attach themselves to the surface of a pathogen and mark it for destruction by other components of the set. The best-known thioester-containing proteins are components C3, C4, and C5 of the mammalian complement system.
Recently, Richard Baxter, working with Yogarany Chelliah and collaborating with Elena Levashina's laboratory (Strasbourg, France), determined the crystal structure of the thioester-containing protein TEP1r from the malaria vector Anopheles gambiae. This protein of about 1,300 amino acids is homologous to the human complement protein C3. In mosquitoes, it is part of the insects' defense against malaria parasites of the species Plasmodium. Levashina and co-workers had found that TEP1 occurs in insects in two different versions whose amino acid sequences are similar (93 percent identical, 96 percent similar). One, called TEP1r, provides 100 percent immunity against a particular strain of Plasmodium; the other, called TEP1s, can kill only 80 percent of the parasites. Mosquitoes with TEP1s in their genome are therefore malaria vectors.
The structure of TEP1r lacks a domain that in human C3 is used for activation by proteolysis. This raises the question of how TEP1r is activated. Most likely, activation also involves a proteolysis event, but a protease responsible for this step in vivo has not yet been found. Another interesting aspect is the difference between the structures of TEP1r and TEP1s. Mapping the sequence of TEP1s on the structure of TEP1r shows that most of the amino acid substitutions between the two proteins are located on the domain that contains the thioester bond. It will be particularly interesting to investigate the effects of these substitutions on interactions between TEP1 and the proteins acting downstream from TEP1. To fill the gaps in our knowledge about insect innate immunity, we plan to continue our collaboration with the Levashina laboratory and to determine structures of other components of this fascinating system.
We also continue our work on bacterial iron transporters, cryptochromes, intramembrane proteases, and various other proteins.