Molecular Chaperone and Transfer Proteins

Molecular Chaperone and Transfer Proteins


Our group is interested in deciphering the molecular principles underlying histone recognition and transfer by histone chaperones, as well as glycosphingolipid and phospholipid recognition and transfer by lipid transfer proteins.


Histone Chaperones

The biology of histone proteins encompasses their synthesis in the cytosol, nuclear import and incorporation into nucleosomes, as well as subsequent eviction from chromatin, redeposition, storage or degradation. Histone chaperones represent a structurally and functionally diverse family of histone-binding proteins that prevent promiscuous interactions of histones before their assembly into chromatin. Our understanding of the mechanisms of histone shuttling between different chaperone systems, and histone transfer onto and off DNA, has been hampered due to the availability of structures of only a limited number of histone-chaperone complexes.

We have written a review on histone chaperones in collaboration with Anja Groth lab (BRIC- Copenhagen).

Hammond, C., Stromme, C. B., Huang, H., Patel, D. J. & Groth, A. (2017). Histone chaperone networks shaping chromatin function. Nat. Rev. Mol. Cell. Biol. 18, 141-158.

DAXX Histone Chaperone. DAXX is a metazoan histone chaperone specific to the evolutionary conserved histone variant H3.3. In collaboration with the David Allis lab (Rockefeller University), we solved the crystal structures of the DAXX histone-binding domain with a histone H3.3-H4 dimer, together with in vitro and in vivo functional studies of structure-guided mutants towards elucidation of the principles underlying H3.3 recognition specificity (Elsasser et al. 2012). DAXX wraps around the H3.3-H4 dimer, with complex formation accompanied by structural transitions in the H3.3-H4 histone fold, thereby competing with major inter-histone, DNA and chaperone ASF1 interaction sites.

In collaboration with the Paul Liebermann lab (University of Pennsylvania), we showed that Epstein-Barr Virus (EBV) tegument BNRF1 is a DAXX-interacting protein required for the establishment of selective viral gene expression during latency (Huang et al. 2016). We solved the structure of BNRF1 DAXX-interaction domain (DID) in complex with DAXX histone-binding domain (HBD) and histones H3.3-H4 that reveal molecular details of virus reprogramming of an anti-viral histone chaperone to promote viral latency and cellular immortalization.

In collaboration with the Peter Lewis lab (Univ. Wisconsin), we solved the x-ray crystal structure of an interaction surface between ATRX and DAXX to an ATRXDAXX complex involved in gene repression and telomere chromatin structure and a DAXXSETDB1KAP1HDAC1 complex that represses endogenous retroviruses independently of ATRX and H3.3 incorporation into chromatin (Hoelper et al. 2017).

Elsasser, S. J., Huang, H., Lewis, P. W., Allis, C. D. & Patel, D. J. (2012). DAXX histone chaperone envelops an H3.3/H4 dimer for H3.3-specific recognition. Nature 491, 560-565.

Huang, H. et al., Lieberman, P. M. & Patel, D. J. (2016). Structural basis underlying viral hijacking of a histone chaperone complex. Nat. Commun. 7: 12707.

Hoelper, D., Huang, H., Jain, A. Patel, D. J. & Lewis, P.W. (2017). Structural and functional insights into ATRX-dependent and -independent functions of the histone chaperone DAXX. Nat. Commun. 8:1193.


MCM2 Histone Chaperone. How histone dynamics are integrated with DNA replication to maintain genome and epigenome information is unclear. We report on a collaborative effort with the Anja Groth lab (BRIC-Copenhagen) on a structure-function analyses showing how human MCM2, a part of the replicative helicase, can chaperone histone H3-H4. Our first structure shows an H3-H4 tetramer bound by two MCM2 histone-binding domains (HBD), hijacking interaction sites used by nucleosomal DNA, while our second structure reveals how MCM2 and ASF1 co-chaperone an H3-H4 dimer (Huang et al. 2015). Further, we demonstrate that MCM2, as part of the MCM2-7 helicase, can chaperone both new and old canonical histones H3-H4, as well as H3.3 and CENPA variants.

Our next structure-function study in collaboration with the Anja Groth lab showed that the homologous recombination (HR) complex TONSL-MMS22L recognizes post-replicative chromatin by binding to H4K20me0 (Saredi et al. 2015). Our structural analysis identifies the TONSL Ankyrin Repeat Domain (ARD) as a histone reader specific for H4K20me0, while H4K20 methylation abrogates TONSL-MMS22L binding. H4K20me0 is a signature of new histones incorporated during DNA replication, demarcating replicated chromatin until cells reach G2/M.

Huang, H. et al., Groth, A. & Patel, D. J. (2015). A unique binding mode enables MCM2 to chaperone histones H3-H4 at replication forks. Nat. Struct. Mol. Biol. 22, 618-626.

Saredi, G., Huang, H. et al., Patel, D. J. & Groth, A. (2016). H4 K20me0 marks post-replicative chromatin and recruits the TONSL-MMS22L DNA repair complex. Nature 534, 714-718.


DNAJC9 Histone Chaperone. From biosynthesis to assembly into nucleosomes, histones are handed through a cascade of histone chaperones, which shield histones from non-specific interactions. Using structure-guided and functional proteomics in collaboration with the Anja Groth and Hongda Huang (SUSTech, Shenzhen) labs, have resulted in identification and characterization of the histone chaperone function of DNAJC9, a heat shock co-chaperone that promotes HSP70-mediated catalysis. We elucidated the structure of DNAJC9, in a histone H3-H4 co-chaperone complex with MCM2, revealing how this dual histone and heat shock co-chaperone binds histone substrates (Hammond et al. 2021). We show that DNAJC9 recruits HSP70-type enzymes via its J domain to fold histone H3-H4 substrates: upstream in the histone supply chain, during replication and transcription-coupled nucleosome assembly, and to clean up spurious interactions.

Hammond, C. M. et al. Patel, D. J., Huang, H. & Groth, A. (2021).  DNAJC9 integrates heat shock molecular chaperones into the histone chaperone network. Mol. Cell 81, 2533-2548.


Lipid Transfer Proteins

Nonenzymic proteins capable of binding lipids exist for a variety of different purposes within cells, including (i) presentation of lipids to hydrolytic proteins for degradation and salvage of breakdown products to rebuild and recycle needed lipid components; (ii) presentation of lipids to proteins of the immune system during development of antigenicity; (iii) sensing of intracellular lipid compositions in membranes of various organelles and (iv) transfer of lipids between intracellular membranes of different organelles. These latter two functions play roles in regulating intracellular signaling events and lipid homeostasis. In collaboration with the Rhoderick Brown lab (Hormel Institute), we have undertaken structure-function studies of glycosphingolipids and phospholipids bound to their lipid transfer proteins.

We have written two reviews on lipid transfer proteins with the Rhoderick Brown lab (Malinina et al. 2015, 2017).

Malinina, L. et al., Patel, D. J. and Brown, R. E. (2015). Sphingolipid transfer proteins defined by the GLTP-fold. Quart. Rev. Biophys. 48, 281-322.

Malinina, L., Patel, D. J. and Brown, R. E. (2017). How alpha-helical motifs form functionally diverse lipid-binding compartments.  Ann. Rev. Biochem. 86, 609-636.


Glycosphingolipid Binding Specificity. The process by which glycosphingolipid GSL-enriched domains are formed, maintained and remodeled are not well defined but are expected to involve specific and highly conserved GSL transfer proteins (GLTPs) that can bind and transfer GSLs between and within cells. In collaboration with the Rhoderick Brown lab, we have solved the crystal structure of 18:1 lactosylceramide bound to GLTP, thereby establishing that the bound GSL is sandwiched, after adaptive recognition, between the alpha-helical layers of the GLTP (Malinina et al. 2004). GSL binding specificity is achieved through recognition and anchoring of the sugar-amide headgroup to the GLTP recognition center by hydrogen-bond networks and hydrophobic contacts, and encapsulation of both lipid chains, in a precisely oriented manner within a ‘molded-to-fit’ hydrophobic tunnel (Malinina et al. 2006). A cleft-like conformational gating mechanism, involving two interhelical loops and one alpha-helix of GLTP, could enable the GSL chains to enter and leave the tunnel in the membrane-associated state (Samygina et al. 2010).

Malinina, L., Malakhova, M. L., Teplov, A., Brown, R. E. & Patel, D. J. (2004). Structural basis for glycosphingolipid transfer specificity. Nature 430, 1048-1053.

Malinina, L., Malakhova, M. L., Kanack, A. T., Brown, R. E. & Patel, D. J. (2006). The liganding mode of glycolipid transfer protein is controlled by glycolipid acyl structure. PLoS Biol. 4, 1996-2011.

Samygina, V. R. et al., Patel, D. J., Brown, R. E., & Malinina, L. (2010). A designer human glycolipid transfer protein with enhanced transfer selectivity for sulfatide. Structure 19, 1644-1654.


Phospholipid Binding Specificity. Phosphorylated sphingolipids ceramide-1-phosphate (C1P) and sphingosine-1-phosphate (S1P) have emerged as key regulators of cell growth, survival, migration and inflammation. C1P produced by ceramide kinase is an activator of group IVA cytosolic phospholipase A2-alpha (cPLA2-alpha), the rate-limiting releaser of arachidonic acid used for pro-inflammatory eicosanoid production, which contributes to disease pathogenesis. In collaboration with the Rhoderick Brown and Charles Chalfont (Virginia Commonwealth University) labs, the lipid transfer protein, CPTP, was shown to specifically transfer C1P between membranes (Simanshu et al. 2013). Crystal structures establish C1P binding through a novel surface-localized, phosphate headgroup recognition center connected to an interior hydrophobic pocket that adaptively expands to ensheath differing-length lipid chains using a cleft-like gating mechanism. These studies have been extended to determination of the structure of phosphatidyl choline bound to the C2-domain of cytosolic phospholipase A2a (Hirano et al. 2019).

Simanshu, D. K. et al., Chalfant, C. E., Brown, R. E. & Patel, D. J. (2013). Nonvesicular trafficking by ceramide-1-phosphate transfer protein regulates eicosanoid production. Nature 500, 463-467.

Hirano, Y. et al., Chalfant, C. E., Patel, D. J. and Brown, R. (2019). Structural basis of phosphatidylcholine recognition by the C2-domain of cytosolic phospholipase A2a. eLife. 8:e44760.