A Heterospecific Leucine Zipper Tetramer

Abstract

Protein-protein interactions play an essential role in the assembly of the macromolecular complexes that form functional networks and control cellular behavior. Elucidating principles of molecular recognition governing potentially complex interfaces is a challenging goal for structural and systems biology. Extensive studies of alpha-helical coiled coils have provided fundamental insight into the determinants of one seemingly tractable class of oligomeric protein interfaces. We report here that two different valine-containing mutants of the GCN4 leucine zipper that fold individually as four-stranded coiled coils associate preferentially in mixtures to form an antiparallel, heterotetrameric structure. X-ray crystallographic analysis reveals that the coinciding hydrophobic interfaces of the hetero- and homotetramers differ in detail, thereby controlling their partnering and structural specificity. Equilibrium disulfide exchange and thermal denaturation experiments show that the 50-fold preference for heterospecificity is determined by interfacial van der Waals interactions and hydrophobicity. Parallel studies of two alanine-containing variants confirm the above-mentioned interpretation of the basis and mechanism of this heterospecificity. Our results suggest that coiled-coil recognition is an inherently geometric process in which heterotypic interaction specificity derives from a complementarity of both shape and chemistry.

Role of positions e and g in the fibrous assembly formation of an amphipathic α‐helix‐forming polypeptide

“…Deng et al examined the structure and stability of GCN4‐pVe, GCN4‐pVg, GCN4‐pAe, and GCN4‐pAg in which either position e or g contained Val or Ala. These polypeptides formed tetramers, and it was shown that the hydrophobic side chain (Val or Ala) at position e or g contributed to one part of the hydrophobic surface with positions a and d .…”

“…Liu et al reported that GCN4‐pAA, a polypeptide that has Ala residues at positions e and g , produces a seven‐stranded coiled‐coil with a cavity within a hydrophobic core . In addition, substitution with Ala or valine (Val) at position g caused increased thermostability compared with the wild‐type amphipathic polypeptide, resulting in the formation of antiparallel tetramers . As mentioned above, it was also reported that the α‐helix assembly state changes depending on whether positions e and g are occupied by charged or hydrophobic amino acids .…”

“…It has been reported from several studies that the assembly state of coiled coils depends on the residues occupying positions e and g , as well as those occupying positions a and d . For example, Alberti et al substituted electrically charged amino acids at positions e and g of a Lac repressor fragment that formed tetrameric coiled‐coils and found that the peptides became dimeric coiled coils .…”

Protein Interaction Module–assisted Function X (PIMAX) Approach to Producing Challenging Proteins Including Hyperphosphorylated Tau and Active CDK5/p25 Kinase Complex

“…Higher ordered interactions could be achieved if trimeric or even tetrameric PIMs were developed. For example, Deng et al reported that two mutant GCN4 leucine zippers form anti-parallel heterospecific tetramers (62). These and similar PIMs might help researchers realize PIMAX applications involving more than two proteins, including enzymes with an obligatory co-factor, and trimeric or tetrameric protein complexes.…”

Stable Cyclohexyl–Phenyl Recognition in the Center of a DNA Duplex

“…Well-known exceptions are, in the case of proteins, hydrophobic interactions of nonpolar amino acid side chains, which can lead to highly ordered structural motifs, such as the leucine zipper, [1,2] and in the case of DNA, the double-helical Watson-Crick structure, in which the two nucleic acid strands are held together by intra-and interstrand aromatic stacking interactions between the nucleobases as the primary source of stability and by hydrogen bonding between the bases as the source of selectivity. Whereas the structural and energetic impact of hydrogen bonding is generally well appreciated, the understanding and prediction of the structural and energetic impact of stacking or hydrophobic interactions on biomolecular folding in water is comparably poor.…”