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Cellular Folding Pathway of a Metastable Serpin

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Cellular Folding Pathway of a Metastable Serpin Cellular folding pathway of a metastable serpin Kshama Chandrasekhara, Haiping Kea, Ning Wanga,b, Theresa Goodwina, Lila M. Gierascha,b,c, Anne Gershensona,b, and Daniel N. Heberta,b,1 aDepartment of Biochemistry and Molecular Biology, University of Massachusetts, Amherst, MA 01003; bProgram in Molecular and Cellular Biology, University of Massachusetts, Amherst, MA 01003; and cDepartment of Chemistry, University of Massachusetts, Amherst, MA 01003 Edited by Armando Parodi, Fundacion Instituto Leloir, Buenos Aries, Argentina, and approved April 28, 2016 (received for review March 1, 2016) Although proteins generally fold to their thermodynamically most The serpin antithrombin III (ATIII) plays an essential role in stable state, some metastable proteins populate higher free energy blood clotting by regulating the activity of thrombin and other states. Conformational changes from metastable higher free energy serine proteases in the coagulation cascade. Numerous misfolding states to lower free energy states with greater stability can then mutations of ATIII are linked to thrombosis (6, 7). The cellular generate the work required to perform physiologically important folding process of ATIII, including its traversal of the secretory functions. However, how metastable proteins fold to these higher pathway, facilitates its folding to the functional metastable high free energy states in the cell and avoid more stable but inactive free energy state. The differing outcomes of unassisted refolding conformations is poorly understood. The serpin family of metastable of purified ATIII and its cellular folding underline the profound protease inhibitors uses large conformational changes that are difference between protein folding reactions in isolation and in downhill in free energy to inhibit target proteases by pulling apart cells and invite further exploration of the key players and steps the protease active site. The serpin antithrombin III (ATIII) targets that make cellular folding so successful (8, 9). The fact that ATIII thrombin and other proteases involved in blood coagulation, and and other serpins must adopt metastable states to function and that ATIII misfolding can thus lead to thrombosis and other diseases. ATIII their misfolding is implicated in several pathologies further raises has three disulfide bonds, two near the N terminus and one near the the importance of understanding their cellular folding pathway. Because disulfide bonding requires two Cys residues to be po- C terminus. Our studies of ATIII in-cell folding reveal a surprising, sitioned within a few angstroms of each other, the intramolecular biased order of disulfide bond formation, with early formation of the disulfide bond reaction is tightly coupled to formation of native C-terminal disulfide, before formation of the N-terminal disulfides, protein topology. Hence, the order of disulfide bond formation critical for folding to the active, metastable state. Early folding of the BIOCHEMISTRY β reveals the progress of its folding; in fact, disulfides have been predominantly -sheet ATIII domain in this two-domain protein con- used as reporters of folding states to map the folding pathway for a strains the reactive center loop (RCL), which contains the protease- N number of proteins both for purified components and in cells binding site, ensuring that the RCL remains accessible. -linked gly- (10–14). ATIII was chosen as a substrate to map the folding path- cans and carbohydrate-binding molecular chaperones contribute to way for serpins as it is one of the few serpin proteins that possess the efficient folding and secretion of functional ATIII. The inability of intramolecular disulfides. Its three disulfide bonds are optimally a number of disease-associated ATIII variants to navigate the folding positioned to report on the evolution of the ATIII structure (Fig. reaction helps to explain their disease phenotypes. S2A). ATIII comprises three β-sheets and nine α-helices combined into two nonsequential, intertwined domains with a central β-sheet cellular protein folding | endoplasmic reticulum | serpins | antithrombin | both forming the core of the α/β domain and bridging the two thrombosis domains. One disulfide, between Cys-8 and Cys-128, links the flexible N terminus to a later helix in the N-terminal, largely helical rreversible switching from one conformation to another allows subdomain; a second, between Cys-21 and Cys-95, connects two Iproteins to perform mechanical work without external energy helical segments that flank the central bridging β-sheet; and the sources such as ATP. Large conformational movements, up to third, between Cys-247 and Cys-430, creates a large loop around A ∼100 Å, can be triggered by proteolysis or changes in environ- the C-terminal third of the molecule (Fig. 1 ). mental conditions, such as pH, initiating processes including membrane fusion for viral infection, protease activation, or in- Significance hibition (1). To facilitate these processes, proteins must fold to kinetically trapped metastable states with relatively high free en- The activity of some proteins requires dramatic conformational ergy. How proteins fold to these states and avoid more thermo- movements. The ability to undergo these movements can be dynamically stable conformations is poorly understood. enabled by folding of these proteins to constrained higher free The serpin family of serine protease inhibitors exemplifies this energy or metastable states. Metastable proteins are particularly type of metastable protein, and its mechanism of folding presents susceptible to misfolding and are therefore associated with a a conundrum. The native, active serpin fold positions the target number of pathologies. Here the cellular folding pathway of the protease-binding site on a loosely structured, accessible stretch serpin antithrombin III (ATIII), which inhibits proteases involved in of sequence termed the reactive center loop (RCL). Once the the coagulation cascade, was determined. ATIII uses a large con- protease forms a covalent acyl intermediate in the scissile bond formational movement in a mousetrap-like mechanism to bind and distort its target protease, resulting in protease inhibition. This in the RCL and cleaves the bond, the serpin undergoes a major work establishes that folding to an active, cocked state requires conformational change like the springing of a mousetrap, and early stabilization of the C-terminal region, which is the last se- the protease is carried ∼70 Å to the opposite side of the serpin, quence translated, explaining how the serpin or mousetrap is set. thereby inactivating the protease by mechanical deformation (2, 3) (Fig. S1). Strikingly, the conformational landscape of serpins Author contributions: N.W., L.M.G., A.G., and D.N.H. designed research; K.C., H.K., N.W., has an alternate fold that is more stable than the functionally and T.G. performed research; K.C., H.K., L.M.G., A.G., and D.N.H. analyzed data; and K.C., required “cocked mousetrap” fold. In this alternative fold, called L.M.G., A.G., and D.N.H. wrote the paper. the latent state, the intact RCL is inserted as an additional strand The authors declare no conflict of interest. into the central β-sheet, resulting in a more stable but inactive This article is a PNAS Direct Submission. state (4). Encoding this gymnastic ability in the folding landscape 1To whom correspondence should be addressed. Email: [email protected]. of serpins comes at a risk: Many mutations in serpins cause mis- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. folding and are associated with diseases called serpinopathies (5). 1073/pnas.1603386113/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1603386113 PNAS Early Edition | 1of6 Downloaded by guest on September 28, 2021 To address the long-standing question of how metastable pro- Secreted ATIII was able to form a covalent complex with thrombin, teins can fold to higher energy states in the cell and avoid lower confirming its successful folding to an active, metastable, and native energy forms, we mapped the folding pathway of ATIII by de- state (see Fig. 3B, lanes 1 and 2). Secreted ATIII was resistant to termining the order in which its disulfides formed. Interestingly, endoglycosidase H (Endo H), which cleaves the high mannose although the C terminus is translated last, it is the first domain to N-glycans attached in the ER but not complex carbohydrates that lock into place. This constraint is key, as it positions the RCL or are added in the Golgi, consistent with its having traversed both the protease bait loop in an exposed orientation that keeps it from ER and Golgi (Fig. 1B). The cellular lysate fraction of ATIII was inserting into an earlier translated sheet where it would reach a distributed between soluble ATIII that was sensitive to Endo H and lower energy but inactive state referred to as the latent state. In- therefore ER-localized (15%) and triton-insoluble protein aggre- terestingly, a number of disease-associated, single-site missense gates that presumably arose from protein that misfolded (10%) mutations in ATIII derail the protein folding program, providing (Fig. 1C). Together these results show that wild-type ATIII is rap- an explanation for some cases of thrombosis. idly and efficiently secreted from CHO cells in a properly folded, native, and active state. Results ATIII Folds Rapidly and Efficiently in Cells. We initially determined The C-Terminal Disulfide of ATIII Forms First and Is Sufficient for the timing of ATIII maturation in live cells. Chinese hamster ovary Secretion. We next dissected the development of native topology (CHO) cells transiently expressing a tagged construct of ATIII in ATIII as it proceeded through the secretory pathway by mon- were pulsed with [35S]-Met/Cys and chased for various times (Fig. itoring the order of formation of its three disulfide bonds. The 1B). Secretion of ATIII into the media occurred with a half-time homogeneously glycosylated N135Q variant of ATIII was used for of 54 min and reached a maximum secretion level of 75% (Fig.
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