De Novo Design of Helical Bundles as Models for Understanding Protein Folding and Function

Protein folding has become one of the best understood biochemical reactions from a kinetic viewpoint. The funneled energy landscape, a consequence of the minimal frustration achieved by evolution in sequences, explains how most proteins fold efficiently and robustly to their functional structure and allows robust prediction of folding kinetics. The folding of Rop (repressor of primer) dimer is exceptional because some of its mutants with a redesigned hydrophobic core both fold and unfold much faster than the WT protein, which seems to conflict with a simple funneled energy landscape for which topology mainly determines the kinetics. We propose that the mystery of Rop folding can be unraveled by assuming a double-funneled energy landscape on which there are two basins that correspond to distinct but related topological structures. Because of the near symmetry of the molecule, mutations can cause a conformational switch to a nearly degenerate yet distinct topology or lead to a mixture of both topologies. The topology predicted to have the lower free-energy barrier height for folding was further found by all-atom modeling to give a better structural fit for those mutants with the extreme folding and unfolding rates. Thus, the non-Hammond effects can be understood within energy-landscape theory if there are in fact two different but nearly degenerate structures for Rop. Mutations in symmetric and regular structures may give rise to frustration and thus result in degeneracy.

Section: Discussionsupporting

confidence: 70%

Symmetry and frustration in protein energy landscapes: A near degeneracy resolves the Rop dimer-folding mystery

Levy

Cho

Shen

et al. 2005

“…The four helices stack together to form a four-helix bundle that is 108 Å long and 20 Å wide. However, the assembly mechanism is distinct from that of many four-helix bundle proteins characterized thus far (21)(22)(23). The HDAC4 four-helix bundle has three perpendicular twofold axes intercepting at the center of the complex.…”

Section: Resultsmentioning

confidence: 93%

Crystal structure of a conserved N-terminal domain of histone deacetylase 4 reveals functional insights into glutamine-rich domains

Guo

Han

Bates

et al. 2007

Glutamine-rich sequences exist in a wide range of proteins across multiple species. A subset of glutamine-rich sequences has been shown to form amyloid fibers implicated in human diseases. The physiological functions of these sequence motifs are not well understood, partly because of the lack of structural information. Here we have determined a high-resolution structure of a glutamine-rich domain from human histone deacetylase 4 (HDAC4) by x-ray crystallography. The glutamine-rich domain of HDAC4 (19 glutamines of 68 residues) folds into a straight ␣-helix that assembles as a tetramer. In contrast to most coiled coil proteins, the HDAC4 tetramer lacks regularly arranged apolar residues and an extended hydrophobic core. Instead, the protein interfaces consist of multiple hydrophobic patches interspersed with polar interaction networks, wherein clusters of glutamines engage in extensive intra-and interhelical interactions. In solution, the HDAC4 tetramer undergoes rapid equilibrium with monomer and intermediate species. Structure-guided mutations that expand or disrupt hydrophobic patches drive the equilibrium toward the tetramer or monomer, respectively. We propose that a general role of glutamine-rich motifs be to mediate protein-protein interactions characteristic of a large component of polar interaction networks that may facilitate reversible assembly and disassembly of protein complexes.amyloid ͉ transcription H istone deacetylases (HDACs) regulate diverse cellular processes through enzymatic deacetylation of both histone and nonhistone proteins (1, 2). Two major classes of this family, class I and class II, share a highly conserved catalytic domain that is targeted by a number of antitumor drugs (3). Class II HDACs, which include HDAC4, HDAC5, and HDAC9, contain an additional N-terminal extension that confers responsiveness to calcium signals and mediates interactions with transcription factors and cofactors (4-7). A prominent feature of this Nterminal region is a highly conserved glutamine-rich domain that can repress transcription independently of the C-terminal catalytic domain (8-10).Glutamine-rich sequences have been found in a variety of eukaryotic proteins, including transcription activators and repressors (11,12). Bioinformatics analysis suggests that glutamine-rich sequences appear to undergo positive selection, suggesting that these low-complexity sequences are conserved for functional reasons (13). Although the physiological functions of most glutamine rich sequences are not well understood, it has been widely observed that high content of glutamines and/or asparagines may lead to increased propensity of amyloid formation implicated in human neurodegenerative diseases and prionlike, non-Mendelian inheritance in yeast (14-16).The intriguing physiological roles of glutamine-rich sequences have attracted much attention to these unusual protein motifs. Max Perutz (15) and others have studied this problem extensively by using x-ray fiber diffraction and modeling. It has been hypothesized that both the...

“…and D.N.W., unpublished data). Thus, deciphering rules that link the sequences of coiled coils to their assembly would have an impact on many aspects of postgenome predictive biology (6) and aid our ability to engineer or even design protein-protein interactions (1,7,8).…”

mentioning

confidence: 99%

Preferred side-chain constellations at antiparallel coiled-coil interfaces

Hadley

Testa

Woolfson

et al. 2008

Reliable predictive rules that relate protein sequence to structure would facilitate postgenome predictive biology and the engineering and de novo design of peptides and proteins. Through a combination of experiment and analysis of the protein data bank (PDB), we have deciphered and rationalized new rules for helixhelix interfaces of a common protein-folding and association motif, the antiparallel dimeric coiled coil. These interfaces are defined by a specific pattern of interactions among largely hydrophobic side chains often referred to as knobs-into-holes (KIH) packing: a knob from one helix inserts into a hole formed by four residues on the partner. Previous work has focused on lateral interactions within the KIH motif, for example, between an a position on one helix and a d position on the other in an antiparallel coiled coil. We show that vertical interactions within the KIH motif, such as a -a-a , are energetically important as well. The experimental and database analyses concur regarding preferred vertical combinations, which can be rationalized as leading to favorable side-chain interactions that we call constellations. The findings presented here highlight an unanticipated level of complexity in coiled-coil interactions, and our analysis of a few specific constellations illustrates a general, multipronged approach to addressing this complexity.backbone thioester exchange ͉ knobs-into-holes packing ͉ peptides ͉ protein design ͉ protein folding