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Photo-crosslinking mass spectrometry localises SNAPC5 and SNAPC2 in SNAPc

ABSTRACT: RNA polymerase III (Pol III) is specialized in the transcription of short, functional RNAs, including small nuclear RNAs (snRNAs). At snRNA genes, Pol III is recruited by the snRNA Activating Protein Complex (SNAPc)forming apreinitiation complex (PIC). Uniquely, SNAPc forms PICs with both Pol II and Pol IIIat their respectivepromoters. Increasingly, snRNA PIC structures arethe focus of structural characterization, however the mechanism of SNAPc cross-polymerase engagementand the role of the SNAPC2 and SNAPC5 subunits in transcription remain unclear. Here, we present CryoEM structuresoftheSNAPc-containing Pol III PIC assembled on the U6 snRNA promoterin the open and melting statesat 3.2-4.2Å resolution. Comparative structural analysis revealedunexpected differences with the corresponding yeast complex and revealedthe basis of SNAPc engagement with both Pol III and Pol IIPICs. Integrating crosslinkingmass spectrometry,we alsolocalizethe SNAPC2 and SNAPC5 subunitsin proximity to the bound promoter DNA, expanding upon existing descriptions of snRNA Pol III PIC structure.

INSTRUMENT(S): Orbitrap Eclipse

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Andrea Graziadei

LAB HEAD: Andrea Graziadei

PROVIDER: PXD053341 | Pride | 2025-01-06

REPOSITORIES: pride

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Source:

			Action	DRS
	240306_SNAPc_FL_contaminants.fasta	Fasta
	240306_Syed_SNAPCDNA_boostBetween_nocons.mzid.gz	Mzid
	240306_Syed_SNAPCDNA_boostBetween_nocons_Links_xiFDR2.1.5.2.csv	Csv
	240306_Syed_SNAPCDNA_boostBetween_nocons_summary_xiFDR2.1.5.2.csv	Csv
	E240306_01_HT_100_Syed_SNAPCflDNA_SDA_SECA5_inj1.raw	Raw

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Project description:[NiFe]-hydrogenases catalyse the reversible splitting of hydrogen. The catalytic metal centre (NiFe(CN)2CO) is unique in biology, and assembled by an intricate protein machinery, in a process that is still being explored. We hypothesised a structural, ATP-dependent, mechanistic explanation for the assembly of the Fe(CN)2CO fragment via the HypCD complex. We carried out a crosslinking mass spectrometry (crosslinking MS) analysis to study the structure of the HypCD complex, both with (holoprotein) and without (apoprotein) the metal cofactor, and both with and without the addition of ATP. We used the UV-photoactivatable crosslinking reagent sulfo-SDA, which has been shown to have excellent performance when studying dynamic and flexible protein complexes. For photoactivation we used a high-powered LED which enabled the use of exceptionally short reaction times (20 seconds) and gave strikingly clear results. From the resulting crosslinked residue pair patterns identified, we were able to unambiguously distinguish between holoprotein with and without ATP. Crosslinks found in holoprotein, in the absence of ATP, suggested a “closed” protein conformation. When the HypCD holoprotein was crosslinked in the presence of ATP, two distinct crosslink bands were almost entirely absent, indicating that the protein conformation had shifted to an “open” conformation. Interestingly, no shift in protein conformation was evident in the crosslinked apoprotein, which implied that the cofactor was central to protein conformational dynamics. Crosslinking MS data helped to explain, and was in agreement with, protein structures predicted by AlphaFold2 (which were subsequently refined by density functional theory (DFT) modeling for placing the cofactor). Considering all the experimental data from this study led to the conclusion that the binding of ATP alone, not its hydrolysis, is required for the transfer of the Fe(CN)2CO fragment to the apo-hydrogenase large subunit.

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Photo-crosslinking mass spectrometry localises SNAPC5 and SNAPC2 in SNAPc

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