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Crystal structure of malaria parasite nucleosome assembly protein: distinct modes of protein localization and histone recognition.

Gill J, Yogavel M, Kumar A, Belrhali H, Jain SK, Rug M, Brown M, Maier AG, Sharma A - J. Biol. Chem. (2009)

Bottom Line: Expression of green fluorescent protein-tagged PfNapL confirmed its exclusive localization to the parasite cytoplasm.A detailed analysis of PfNapL structure suggests unique histone binding properties.The crucial structural differences observed between parasite and yeast NAPs shed light on possible new modes of histone recognition by nucleosome assembly proteins.

View Article: PubMed Central - PubMed

Affiliation: Structural and Computational Biology Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Road, New Delhi 110067, India.

ABSTRACT
Nucleosome assembly proteins (NAPs) are histone chaperones that are essential for the transfer and incorporation of histones into nucleosomes. NAPs participate in assembly and disassembly of nucleosomes and in chromatin structure organization. Human malaria parasite Plasmodium falciparum contains two nucleosome assembly proteins termed PfNapL and PfNapS. To gain structural insights into the mechanism of NAPs, we have determined and analyzed the crystal structure of PfNapL at 2.3 A resolution. PfNapL, an ortholog of eukaryotic NAPs, is dimeric in nature and adopts a characteristic fold seen previously for yeast NAP-1 and Vps75 and for human SET/TAF-1b (beta)/INHAT. The PfNapL monomer is comprised of domain I, containing a dimerization alpha-helix, and a domain II, composed of alpha-helices and a beta-subdomain. Structural comparisons reveal that the "accessory domain," which is inserted between the domain I and domain II in yeast NAP-1 and other eukaryotic NAPs, is surprisingly absent in PfNapL. Expression of green fluorescent protein-tagged PfNapL confirmed its exclusive localization to the parasite cytoplasm. Attempts to disrupt the PfNapL gene were not successful, indicating its essential role for the malaria parasite. A detailed analysis of PfNapL structure suggests unique histone binding properties. The crucial structural differences observed between parasite and yeast NAPs shed light on possible new modes of histone recognition by nucleosome assembly proteins.

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Two views of the surface representation of PfNapL and yNAP-1 dimers. Corresponding residues from the mutagenesis data of hSET are colored green and red. The identical/conserved surface residues within the NAP family are colored yellow.
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fig7: Two views of the surface representation of PfNapL and yNAP-1 dimers. Corresponding residues from the mutagenesis data of hSET are colored green and red. The identical/conserved surface residues within the NAP family are colored yellow.

Mentions: Prediction of Essential Residues Involved in Histone Binding by PfNapL—Most histone chaperones contain long acidic stretches in their C-terminal region, which were thought to be involved in histone binding, considering the highly basic character of histones. However, recent studies have shown that these acidic stretches are not essential for histone binding activities for chaperones yNAP-1, Asf1, nucleoplasmin, etc. (5, 15, 37). It is noted that full-length PfNapL also has an acidic stretch of residues in its C-terminal region similar to yNAP-1 and hSET, with an average pI of ∼3.0 calculated using amino acid sequence (Fig. 1a). Mutagenesis studies on hSET revealed important residues of domain II that affect the binding of hSET to both core histones and double-stranded DNA (16). We have mapped these residues on yNAP-1 and PfNapL (Fig. 6a). Based upon these hSET mutagenesis data, we identified several corresponding residues in PfNapL and mutated them to test their relevance in histone recognition. We constructed six single-site mutants of the corresponding residues from PfNapL (D192A, H227A, T230A, Y259A, E260A, and K266A). In addition, we made one other mutant (D223A) that is conserved, exposed, and proximal to the previously implicated histone recognition residues from hSET (16). Our PfNapL-histone binding data, using purified proteins of high quality, show that none of these seven mutations altered histone binding of PfNapL to histones H3, H4, H2A, or H2B, to histone tetramer, or to histone octamer significantly (Fig. 6b). We therefore propose that it is likely for histones to have different a binding site(s) on PfNapL (and possibly on other NAPs) in comparison with hSET-histone interactions (16). These residues are not identical among PfNapL, hSET, yNAP-1, and Vps75 proteins, and in general, residues proposed by hSET-histone studies show weak conservation (Table S1). An overall structure-based residue comparison between yNAP-1, Vps75, hSET, and PfNapL reveals very few regions of sequence conservation (Fig. S1). In order to further probe the potential sites for histone recognition by PfNapL, we inspected the surface residues of PfNapL (keeping in view structure and sequence information from NAPs). Here, we have highlighted the identical/conserved surface residues in the NAP family (Figs. 1d and 7) based on the described criterion.


Crystal structure of malaria parasite nucleosome assembly protein: distinct modes of protein localization and histone recognition.

Gill J, Yogavel M, Kumar A, Belrhali H, Jain SK, Rug M, Brown M, Maier AG, Sharma A - J. Biol. Chem. (2009)

Two views of the surface representation of PfNapL and yNAP-1 dimers. Corresponding residues from the mutagenesis data of hSET are colored green and red. The identical/conserved surface residues within the NAP family are colored yellow.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2665062&req=5

fig7: Two views of the surface representation of PfNapL and yNAP-1 dimers. Corresponding residues from the mutagenesis data of hSET are colored green and red. The identical/conserved surface residues within the NAP family are colored yellow.
Mentions: Prediction of Essential Residues Involved in Histone Binding by PfNapL—Most histone chaperones contain long acidic stretches in their C-terminal region, which were thought to be involved in histone binding, considering the highly basic character of histones. However, recent studies have shown that these acidic stretches are not essential for histone binding activities for chaperones yNAP-1, Asf1, nucleoplasmin, etc. (5, 15, 37). It is noted that full-length PfNapL also has an acidic stretch of residues in its C-terminal region similar to yNAP-1 and hSET, with an average pI of ∼3.0 calculated using amino acid sequence (Fig. 1a). Mutagenesis studies on hSET revealed important residues of domain II that affect the binding of hSET to both core histones and double-stranded DNA (16). We have mapped these residues on yNAP-1 and PfNapL (Fig. 6a). Based upon these hSET mutagenesis data, we identified several corresponding residues in PfNapL and mutated them to test their relevance in histone recognition. We constructed six single-site mutants of the corresponding residues from PfNapL (D192A, H227A, T230A, Y259A, E260A, and K266A). In addition, we made one other mutant (D223A) that is conserved, exposed, and proximal to the previously implicated histone recognition residues from hSET (16). Our PfNapL-histone binding data, using purified proteins of high quality, show that none of these seven mutations altered histone binding of PfNapL to histones H3, H4, H2A, or H2B, to histone tetramer, or to histone octamer significantly (Fig. 6b). We therefore propose that it is likely for histones to have different a binding site(s) on PfNapL (and possibly on other NAPs) in comparison with hSET-histone interactions (16). These residues are not identical among PfNapL, hSET, yNAP-1, and Vps75 proteins, and in general, residues proposed by hSET-histone studies show weak conservation (Table S1). An overall structure-based residue comparison between yNAP-1, Vps75, hSET, and PfNapL reveals very few regions of sequence conservation (Fig. S1). In order to further probe the potential sites for histone recognition by PfNapL, we inspected the surface residues of PfNapL (keeping in view structure and sequence information from NAPs). Here, we have highlighted the identical/conserved surface residues in the NAP family (Figs. 1d and 7) based on the described criterion.

Bottom Line: Expression of green fluorescent protein-tagged PfNapL confirmed its exclusive localization to the parasite cytoplasm.A detailed analysis of PfNapL structure suggests unique histone binding properties.The crucial structural differences observed between parasite and yeast NAPs shed light on possible new modes of histone recognition by nucleosome assembly proteins.

View Article: PubMed Central - PubMed

Affiliation: Structural and Computational Biology Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Road, New Delhi 110067, India.

ABSTRACT
Nucleosome assembly proteins (NAPs) are histone chaperones that are essential for the transfer and incorporation of histones into nucleosomes. NAPs participate in assembly and disassembly of nucleosomes and in chromatin structure organization. Human malaria parasite Plasmodium falciparum contains two nucleosome assembly proteins termed PfNapL and PfNapS. To gain structural insights into the mechanism of NAPs, we have determined and analyzed the crystal structure of PfNapL at 2.3 A resolution. PfNapL, an ortholog of eukaryotic NAPs, is dimeric in nature and adopts a characteristic fold seen previously for yeast NAP-1 and Vps75 and for human SET/TAF-1b (beta)/INHAT. The PfNapL monomer is comprised of domain I, containing a dimerization alpha-helix, and a domain II, composed of alpha-helices and a beta-subdomain. Structural comparisons reveal that the "accessory domain," which is inserted between the domain I and domain II in yeast NAP-1 and other eukaryotic NAPs, is surprisingly absent in PfNapL. Expression of green fluorescent protein-tagged PfNapL confirmed its exclusive localization to the parasite cytoplasm. Attempts to disrupt the PfNapL gene were not successful, indicating its essential role for the malaria parasite. A detailed analysis of PfNapL structure suggests unique histone binding properties. The crucial structural differences observed between parasite and yeast NAPs shed light on possible new modes of histone recognition by nucleosome assembly proteins.

Show MeSH
Related in: MedlinePlus