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. 2002 Oct;11(10):2471-8.
doi: 10.1110/ps.0212602.

A structure for the yeast prohibitin complex: Structure prediction and evidence from chemical crosslinking and mass spectrometry

Jaap W Back  1 , Marta Artal Sanz, Luitzen De Jong, Leo J De Koning, Leo G J Nijtmans, Chris G De Koster, Les A Grivell, Hans Van Der Spek, Anton O Muijsers
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A structure for the yeast prohibitin complex: Structure prediction and evidence from chemical crosslinking and mass spectrometry

Jaap W Back et al. Protein Sci. 2002 Oct.

Abstract

The mitochondrial prohibitin complex consists of two subunits (PHB1 of 32 kD and PHB2 of 34 kD), assembled into a membrane-associated supercomplex of approximately 1 MD. A chaperone-like function in holding and assembling newly synthesized mitochondrial polypeptide chains has been proposed. To further elucidate the function of this complex, structural information is necessary. In this study we use chemical crosslinking, connecting lysine side chains, which are well scattered along the sequence. Crosslinked peptides from protease digested prohibitin complexes were identified with mass spectrometry. From these results, spatial restraints for possible protein conformation were obtained. Many interaction sites between PHB1 and PHB2 were found, whereas no homodimeric interactions were observed. Secondary and tertiary structural predictions were made using several algorithms and the models best fitting the spatial restraints were selected for further evaluation. From the structure predictions and the crosslink data we derived a structural building block of one PHB1 and one PHB2 subunit, strongly intertwined along most of their length. The size of the complex implies that approximately 14 of these building blocks are present. Each unit contains a putative transmembrane helix in PHB2. Taken together with the unit building block we postulate a circular palisade-like arrangement of the building blocks projecting into the intermembrane space.

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Figures

Fig. 1.
Fig. 1.
Reelectrophoresis of the electroeluted PHB complex. The PHB complex was cut out of a first dimension BNE gel and electroeluted as described in Materials and Methods. (A) Control incubation of an aliquot of complex in crosslink buffer for the same duration as a crosslinking experiment. After incubation the complex was run again on a native first dimension and subsequently on a denaturing (SDS) second dimension. Protein was visualized by an anti-PHB1 antibody. Approximately 90% of the loaded amount of protein is still in intact complexes running at 1 MD. (B) Crosslinking with sBID. An aliquot of the electroeluted complex was incubated with 1 mM sBID as described in Materials and Methods. Western analysis after BNE2D revealed bands running at 1 MD in the first dimension (size of the PHB complex) that dissociated into 70-kD bands in the second dimension (size of PHB dimers).
Fig. 2.
Fig. 2.
Aligned sequences of PHB1 and PHB2. The region expected to be membrane spanning in PHB2 and membrane associated in PHB1 is underlined. Lysine residues (possible _targets of the crosslinkers used in this study) are boxed. It can be seen that lysines are well distributed along the chains.
Fig. 3.
Fig. 3.
ESI-QTOF-MSMS analysis of a DTSP crosslinked peptide. P1K204 is linked to P2K233. The singly charged peptide was seen in MALDI-TOF at m/z 2398.2 (deviation from calculated mass = 14 ppm). In ESI a triply charged ion was seen at m/z 800.1 and fragmented by low-energy CID as described in Materials and Methods. (A) Structure of the crosslinked peptide: the lysine residues are linked by their ɛ-amino groups through DTSP. Observed fragment ions are indicated (Roepstorff and Fohlman nomenclature). (B) ESI-QTOF-MSMS spectrum: fragment ions corresponding to the ions marked in (A) are annotated.
Fig. 4.
Fig. 4.
Predicted secondary structure of the PHB proteins. Solid blocks indicate α-helices, filled arrows indicate β-sheets. Alignment is the same as in Figure 2 ▶. Crosslinks found in this study (see also Table 1) are indicated. Most crosslinks connect residues in PHB1 to residues in PHB2.
Fig. 5.
Fig. 5.
The model of a unit-cell building block. An elongated three segment assembly consisting of a membrane anchor, a central β-sheet and a protruding four α-helix bundle (similar to the four helix-bundles of Snare proteins) is postulated.
Fig. 6.
Fig. 6.
A representation of the superstructure of the complex. The membrane associated elongated building blocks assemble in a ring shaped structure, probably acting as a holdase/unfoldase for newly synthesized mitochondrial proteins. (A) Dimeric building block (top view). (B) Proposed circular arrangement of the building blocks (top view). (C) A section of four building blocks and the mitochondrial inner membrane (side view).
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References

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