Interfacial binding and aggregation of lamin A tail domains associated with Hutchinson–Gilford progeria syndrome
Graphical abstract
Introduction
Many human diseases are caused by mutations that alter protein structure and thereby function. In most cases, altered protein structure reduces or eliminates function leading to disease pathology. However, altered protein structures can also lead to interfacial mislocalization, aggregation and other architectural changes. Hutchinson Gilford Progeria Syndrome (HGPS) is an accelerated aging disorder caused by a single base pair mutation (DNA C1824T causes G608G) in LMNA, the gene coding for lamin A [1]. The DNA mutation activates a cryptic splice site, and the resulting mutant protein, ∆50 lamin A (Δ50LA), lacks 50 amino acids in its tail domain and as a consequence retains the branched lipid farnesyl group which is added to the C-terminus during posttranslational processing.
HGPS cells show a thicker, stiffer nucleoskeleton with reduced filament exchange [2], [3]. The increased association of Δ50LA with the nuclear membrane also affects processes within the nucleus such as DNA repair and transcription [4] as well as transport through nuclear pores [5]. The hyper-association of Δ50LA and other farnesylated lamin mutants with the nuclear membrane is thought to result from the retained lipidation [6], similar to other farnesylated proteins such as Ras and Rho proteins, which show preferential localization to the plasma membrane [7], [8]. However, the farnesyl group alone provides only weak protein–membrane association [9], and other factors such as charged amino acid clusters on the protein's membrane-binding interface, a second lipid group or altered binding to transmembrane proteins are likely required for stable protein–membrane association [10].
The primary accepted hypothesis is that the farnesyl lipidation of the Δ50LA is responsible for the pathology of HGPS, including using farnesyl transferase inhibitors as treatment options for patients [11], [12]. Here, we suggest that the interaction of LA with the membrane and the hyper-interaction of mutant Δ50LA with the membrane may also be enhanced by electrostatic interactions and aggregation. To this end, we quantify binding of the C-terminal tail domain (TD) of recombinant LA and farnesylated LA variants to synthetic membrane models, sparsely-tethered bilayer lipid membranes (stBLMs). The TDs are spatially distinct from the adjacent rod domains, which are responsible for filament assembly [13], [14]. We find that the ∆50LA-TD forms aggregates or complexes at the membrane interface, both in the unfarnesylated and farnesylated forms, whereas the mature wild type LA-TD (mwtLA-TD) does not exceed a monolayer of protein at the membrane interface. Thus, the studies of a well-defined synthetic model system reported here suggests a mechanism by which Δ50LA may trigger the formation of a thicker, [3] stiffer [2] nucleoskeleton with altered microdomain structures [2] that accumulate and resist proteolysis [15].
Section snippets
Protein expression, purification and modification
Δ50LA-TD and mwtLA-TD were expressed and purified from recombinant plasmids as described previously [16]. In brief, the TD portion (from R386 to the C-terminus) was co-expressed with GST in BL21 Codon-Plus cells (Agilent) at 37 °C. Purification was performed with glutathione magnetic beads (Pierce) and the protein was cleaved enzymatically with proTEV cleavage enzyme (Promega) at 30 °C for 5–7 h. The cleaved protein was further purified by exposure to agarose glutathione beads (Pierce) to remove
mwtLA-TDs form a single layer on stBLM at low ionic strength
We previously showed the membrane association of the farnesylated ∆50LA-TD, which was expected given the covalently bound lipid group [16]. Here, we examine the membrane association of mwtLA-TD, which lacks farnesylation in its mature form within the nucleoskeleton of the cell. Protein was titrated onto a DOPC:DOPS (95:5) stBLM in 50 mM HEPES at pH 7.2, which we refer to as ‘low ionic strength’ in this paper (Fig. 1). Fitting the data to a Langmuir model yields an equilibrium dissociation
Discussion
We demonstrated experimentally and with all-atom MD simulations in previous work that Ca2 + induces a conformational change in the tail domains of prelamin A and ∆50 lamin A that exposes the farnesyl group to the exterior of the protein [17]. Here, we observed that fn-∆50LA-TD is stable in Mg2 + but aggregated in the presence of Mn2 + and Zn2 +. The significance of fn-∆50LA-TD aggregation in the presence of Mn2 + and Zn2 + is unknown, but both ions are found as co-factors for enzymes inside the
Conclusion
In this work, the membrane affinities of LA-TD and its pathogenic mutants provide insight in the toxic enhancement of ∆50LA with the inner nuclear membrane. We find that both the normal mwtLA and unfarnesylated mutant ∆50LA-TD can associate with charged membranes in low ionic strength buffer, but ∆50LA-TD exceeds a monolayer on the membrane surface. The farnesylated form of ∆50LA-TD binds the membrane strongly at physiological levels of salt in the presence of Ca2 + or Mg2 +, but membrane
Acknowledgments
We acknowledge the help in protein production and purification by Matthew Biegler (CMU MSE) and Kelli Coffey (CMU ChemE) and funding received by K.N.D. (Progeria Research Foundation and NSF CBET — 0954421 CAREER), A.K. (NIH-NIA NRSA F30-AG030905) and M.L. (NIH-NIGMS 1R01-GM101647). M.J.B. and Z.Q. acknowledge support from ONR-PECASE N000141010562 and NIH U01 EB014976.
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