Human heat shock protein 70 (Hsp70) as a peripheral membrane protein

Abstract

While a significant fraction of heat shock protein 70 (Hsp70) is membrane associated in lysosomes, mitochondria, and the outer surface of cancer cells, the mechanisms of interaction have remained elusive, with no conclusive demonstration of a protein receptor. Hsp70 contains two Trps, W90 and W580, in its N-terminal nucleotide binding domain (NBD), and the C-terminal substrate binding domain (SBD), respectively. Our fluorescence spectroscopy study using Hsp70 and its W90F and W580F mutants, and Hsp70-∆SBD and Hsp70-∆NBD constructs, revealed that binding to liposomes depends on their lipid composition and involves both NBD and SBD. Association of Hsp70 with phosphatidylcholine (PC) liposomes is weak, with insertion of its Trps into the bilayer hydrocarbon region. In the presence of cardiolipin (CL), bis-monoacylglycero phosphate (BMP), or phosphatidylserine (PS) Hsp70 attaches to membranes peripherally, without penetration. Our data suggest that the organelle distribution of Hsp70 is determined by their specific lipid compositions, with Hsp70 associating with the above lipids in mitochondria, lysosomes, and the surface of cancer cells, respectively. NBD and SBD attach to lipids by extended phospholipid anchorage, with specific acidic phospholipids associating with Hsp70 in the extended conformation with acyl chains inserting into hydrophobic crevices within Hsp70, and other chains remaining in the bilayer. This anchorage is expected to cause a stringent orientation of Hsp70 on the surface. Our data further suggest that acidic phospholipids induce a transition of SBD into the molten globule state, which may be essential to allow SBD-substrate interaction also within the hydrophobic bilayer interior acyl chain region.

Biochemical characterization of the interaction between HspA1A and phospholipids

“…Although these results are congruent with previous reports on different Hsp70s showing that both protein domains are important for the association of the chaperone with lipids and this association is lipid-specific (Mamelak and Lingwood 2001;) Mamelak and Lingwood 2001;Sahu et al 2011) (Armijo et al 2014;Harada et al 2007Harada et al , 2014Harada et al , 2015Mahalka et al 2014), they do not directly show which part of the protein embeds in the lipid bilayer. However, Armijo et al (2014) showed that in the case of PS, the SBD region of HspA1A embeds within the lipid bilayer.…”

“…This initial report was followed by many investigators showing that several members of the Hsp70 protein family, including HspA1A, as well as several bacterial Hsp70 (DnaK) proteins interact specifically with multiple glycolipids and phospholipids. These include phosphatidylserine (PS), bis-(monoacylglycero)-phosphate (BMP), globoyltriaosyl-ceramide (Gb3), sulfo-glycolipids, and anandamide (Arispe and De Maio 2000;Arispe et al 2002Arispe et al , 2004Armijo et al 2014;Broquet et al 2003;Browne et al 2007;Chen et al 2005;Gehrmann et al 2008;Harada et al 2007Harada et al , 2014Harada et al , 2015Kirkegaard et al 2010;Mahalka et al 2014;McCallister et al 2015;Oddi et al 2009;Petersen et al 2010;Whetstone and Lingwood 2003). These reports demonstrate that, although Hsp70s do not contain known lipid-binding domains, they interact with specific lipids and this interaction is evolutionarily conserved.…”

“…Lipidspecific binding is also supported by the different theoretical binding models (McCallister et al 2015) and the lipidspecific differences observed by us (Figs. 2b,5b,and 6b) and others (Armijo et al 2014;Harada et al 2014Harada et al , 2015Mahalka et al 2014;Mamelak and Lingwood 2001;. Arispe and De Maio (2000) and later Armijo et al (2014) showed that HspA1A embeds in the lipid bilayer of PS liposomes.…”

“…However, several observations argue against this suggestion. First, both aromatic and positive amino acids have been implicated in the association between Hsp70s with BMP, PS, and SGC Mahalka et al 2014;Mamelak and Lingwood 2001;Sahu et al 2011). Second, HspA1A embeds in the lipid bilayer composed of PS, PA, PG [ Fig.…”

“…These reports revealed that the binding of HspA1A to PS and other lipids depends on the lipid environment and that the protein is most probably using different mechanisms and different regions to bind to particular lipids (Arispe et al 2004;Armijo et al 2014;Harada et al 2007Harada et al , 2015Kirkegaard et al 2010;Mahalka et al 2014;Mamelak and Lingwood 2001;McCallister et al 2015;Schilling et al 2009;Whetstone and Lingwood 2003). Yet, the molecular mechanism of the interaction between HspA1A and different anionic lipids remains elusive.…”