Dataset Information

Structural analysis of human cis-prenyltransferase

ABSTRACT: Isoprenoids are synthesized by the prenyltransferases superfamily, which is subdivided according to the product stereoisomerism and length. In short- and medium-chain isoprenoids, product length correlates with active site volume. However, enzymes synthesizing long-chain products and rubber synthases fail to conform to this paradigm, due to an unexpectedly small active site. Here, we focused on the human cis-prenyltransferase complex (hcis-PT), residing at the endoplasmic reticulum membrane and playing a crucial role in protein glycosylation. Crystallographic investigation of hcis-PT along the reaction cycle revealed an outlet for the elongating product. Hydrogen-deuterium exchange mass spectrometry analysis showed that the hydrophobic active site core is flanked by dynamic regions consistent with separate inlet and outlet orifices. Finally, using a fluorescence substrate analog, we show that product elongation and membrane association are closely correlated. Together, our results support direct membrane insertion of the elongating isoprenoid during catalysis, uncoupling active site volume from product length.

INSTRUMENT(S): Bruker Daltonics solarix series

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Petr Man

LAB HEAD: Petr Man

PROVIDER: PXD031707 | Pride | 2022-06-09

REPOSITORIES: pride

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Structural basis for long-chain isoprenoid synthesis by cis-prenyltransferases.

Giladi Moshe M Lisnyansky Bar-El Michal M Vaňková Pavla P Ferofontov Alisa A Melvin Emelia E Alkaderi Suha S Kavan Daniel D Redko Boris B Haimov Elvira E Wiener Reuven R Man Petr P Haitin Yoni Y

Science advances 20220518 20

Isoprenoids are synthesized by the prenyltransferase superfamily, which is subdivided according to the product stereoisomerism and length. In short- and medium-chain isoprenoids, product length correlates with active site volume. However, enzymes synthesizing long-chain products and rubber synthases fail to conform to this paradigm, because of an unexpectedly small active site. Here, we focused on the human cis-prenyltransferase complex (hcis-PT), residing at the endoplasmic reticu ...[more]

PMID: 35584224

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Project description:Post expression from the host cell, a bio-therapeutic will be subjected to several downstream processing steps prior to final formulation. Analysis of the product under various buffer conditions at each stage is desirable to show that the integrity of the sample is maintained and to monitor any potential product loss. In this study we examine how varying buffer conditions during downstream processing impact the structural integrity of a biopharmaceutical product, with a specific focus on the model IgG1 antibody, mAb4, provided by FUJIFILM Diosynth Biotechnologies. Flexibility, stability, aggregation propensity, and bulk properties were evaluated across the buffers of four sample points, including perfusion media, purification stages, and formulation buffer. Comparisons with Herceptin, an extensively studied IgG1 antibody, were conducted in a mass spectrometry-compatible buffer. Although mAb4 and Herceptin present with similar charge state distributions (CSD) in native mass spectrometry (MS) experiments, they exhibit distinct unfolding patterns during the activated ion mobility-mass spectrometry (aIM-MS) and differential scanning fluorimetry (DSF). The greater structural stability and aggregation onset temperature (Tagg) observed in Herceptin, are primarily attributed to the heavier glycosylation and kappa-class light chains present in Herceptin compared to the lambda-class light chains in mAb4. Hydrogen deuterium exchange mass spectrometry (HDX-MS) revealed that while mAb4 experiences substantial structural changes during purification, characterized by high flexibility, a low melting temperature (Tm), and prevalent repulsive protein-protein interactions, it eventually transitions to a highly compact and stable structure in buffers with higher salt concentrations and during formulation. Notably, in the formulated environment, the third constant domain (CH3) of the heavy chain retains flexibility and is identified as a region of interest for aggregation formation. Here we showcase the integration of MS and orthogonal techniques to attain comprehensive information which can be utilized early in the development stage to aid in decision-making regarding targeted mutations or to guide the design space of bioprocesses and formulation choices.

			Action	DRS
	DHDDS.fasta	Fasta
	NBGR.fasta	Fasta
	PM_cisPT-crap_20210603.fasta	Fasta
	checksum.txt	Txt
	cisPT_10m_12262.d.zip	Other

Dataset Information

Structural analysis of human cis-prenyltransferase

Dataset's files

Publications

Structural basis for long-chain isoprenoid synthesis by <i>cis</i>-prenyltransferases.

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