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Drosophila melanogaster Heterochromatin Protein 1a (HP1a) is essential for compacted heterochromatin structure and associated gene silencing. Its chromo shadow domain (CSD) is well-known for binding to peptides that contain a PXVXL motif. Heterochromatin protein 2 (HP2) is a nonhistone chromosomal protein that associates with HP1a in the pericentric heterochromatin, telomeres and the fourth chromosome. Using NMR spectroscopy, fluorescence polarization and site-directed mutagenesis, we identified an LCVKI motif in HP2 that binds to the HP1a CSD. The binding affinity of the HP2 fragment is approximately two orders of magnitude higher than that of peptides from PIWI (with a PRVKV motif), AF10 (with a PLVVL motif), or CG15356 (with LYPLL and LSIVA motifs). To delineate differential interactions of the HP1a CSD, we characterized its structure, backbone dynamics and dimerization constant. We find that the dimerization constant is bracketed by the affinities of HP2 and PIWI, which dock to the same HP1a homodimer surface. This suggests that HP2, but not PIWI, interaction can drive homodimerization of HP1a. Interestingly, the integrity of the disordered C-terminal extension (CTE) of HP1a is essential for discriminatory binding, whereas swapping the PXVXL motifs does not confer specificity. Serine phosphorylation at the peptide binding surface of the CSD is thought to regulate heterochromatin assembly. Glutamic acid substitution at these sites destabilizes HP1a dimers, but improves the interaction with both binding partners. Our studies underscore the importance of CSD dimerization and cooperation with the CTE in forming distinct complexes of HP1a.

Cytochromes c and c1are heme proteins that are essential for aerobic respiration. Release of cytochrome c from mitochondria is an important signal in apoptosis initiation. Biogenesis of c-type cytochromes involves covalent attachment of heme to two cysteines (at a conserved CXXCH sequence) in the apocytochrome. Heme attachment is catalyzed in most mitochondria by holocytochrome c synthase (HCCS), which is also necessary for import of apocytochrome c. Thus, HCCS affects cellular levels of cytochrome c, impacting mitochondrial physiology and cell death. Here, we review the mechanisms of HCCS function and the roles played by heme and residues in the CXXCH motif. Additionally, we consider concepts emerging within the two prokaryotic cytochrome c biogenesis pathways.

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Cytochrome c (cyt c), required for electron transport in mitochondria, possesses a covalently attached heme cofactor. Attachment is catalyzed by holocytochrome c synthase (HCCS), leading to two thioether bonds between heme and a conserved CXXCH motif of cyt c. In cyt c, histidine (His19) of CXXCH acts as an axial ligand to heme iron and upon release of holocytochrome c from HCCS, folding leads to formation of a second axial interaction with methionine (Met81). We previously discovered mutations in human HCCS that facilitate increased biosynthesis of cyt c in recombinant Escherichia coli. Focusing on HCCS E159A, novel cyt c variants in quantities that are sufficient for biophysical analysis are biosynthesized. Cyt c H19M, the first bis-Met liganded cyt c, is compared with other axial ligand variants (M81A, M81H) and single thioether cyt c variants. For variants with axial ligand substitutions, electronic absorption, near-UV circular dichroism, and electron paramagnetic resonance spectroscopy provide evidence that axial ligands are changed and the heme environment is altered. Circular dichroism spectra in far UV and thermal denaturation analyses demonstrate that axial ligand changes do not affect secondary structures and stability. Redox potentials span a 400-mV range (+349 mV vs. standard hydrogen electrode, H19M; +252 mV, WT; −19 mV, M81A; −69 mV, M81H). We discuss the results in the context of a four-step mechanism for HCCS, whereby HCCS mutants such as E159A are enhanced in release (step 4) of cyt c from the HCCS active site; thus, we term these “release mutants.”