Four helix bundle motif

Two such dimers form the tetramer through mainly hydrophobic interactions between the a helices. The p strands are on the outside of the tetramer and are not involved in the dimer-dimer interactions. The arrangement of the four a helices is unusual and provides a rare example of four a helices packed against each other in a way different from the four-helix bundle motif. 

Alpha helices D and E from the L and M subunits (Figure 12.14) form the core of the membrane-spanning part of the complex. These four helices are tightly packed against each other in a way quite similar to the four-helix bundle motif in water-soluble proteins. Each of these four helices provides a histidine side chain as ligand to the Ee atom, which is located between the helices close to the cytoplasm. The role of the Ee atom is probably to... 

The structurally similar L and M subunits are related by a pseudo-twofold symmetry axis through the core, between the helices of the four-helix bundle motif. The photosynthetic pigments are bound to these subunits, most of them to the transmembrane helices, and they are also related by the same twofold symmetry axis (Figure 12.15). The pigments are arranged so that they form two possible pathways for electron transfer across the membrane, one on each side of the symmetry axis. 

Helical heptad repeat sequences have been reported to be well behaved although they are difficult to characterize by NMR spectroscopy due to spectral overlap. The motifs that have been shown to have native-like properties, and are not highly repetitive, have cores composed of aromatic amino acid side chains of, for example, phenylalanine and tryptophan. In four-helix bundle motifs [1, 2], the /1/la-motif BBAl [5] and the /1-sheet protein Betanova [9], the formation of the folded structure appears to be strongly dependent on such residues although the energetics have not been calculated by substitution studies. As a tentative rule, therefore, the probability of success in the design of a new protein is probably much higher if residues are included that can form aromatic clusters in the core (Fig. 5). 

Fig. 14. Reaction mechanism of histidine-catalyzed acyl transfer of reactive esters. The excised reactive site is part of a four-helix bundle motif and is capable of substrate recognition and rate enhancements of approximately three orders of magnitude...

The defining structural feature of the hematopoietic class of cytokines is a four-helix bundle motif organized into four anti-parallel hehces that adopt an up-up-down-down motif (Bazan, 1990a Sprang and Bazan,... 

Helical regions predicted from the sequence of E. coli BFR (85) are approximately 6-34, 36-65. 78-105.115-143. and 150-157, and a possible four-helix bundle motif is shown in Fig. 10. 

The three-dimensional structure of CNTF revealed a four helix bundle motif (heliees A to D MeDonald et al.,... 

The iron transport and storage proteins transferrin and ferritin (Figures 6 and 7) are responsible for iron homeostasis in organisms after uptake e.g. through the intestine. Ferritins are assemblies of 24 polypeptide subunits, each of which has a four-helix-bundle motif. This protein directs the oxidation of Fe(II) (ferroxidase activity), the translocation of Fe(II) and Fe(III) and the mineralization of iron in the inner core of ferritin. Structures are available from, e.g., vertebrates and bacteria. The latter have two forms of ferritin, one related to the vertebrate structures discussed above, the other comprising iron mainly in the form of heme in addition, there is a dinuclear metal-binding site denoted the ferroxidase site, where the iron binds for oxidation. 

Figure 8.22 The lac repressor molecule is a V-shaped tetramer in which each arm is a dimer containing a DNA-hinding site. The helix-tum-helix motifs (red) of each dimer bind in two successive major grooves and the hinge helices (purple) bind adjacent to each other in the minor groove between the two major groove binding sites. The four subunits of the tetramer are held together by the four C-terminal helices (yellow) which form a four helix bundle. The bound DNA fragments are bent. (Adapted from M. Lewis et al., Science 271 1247-1254, 1996.)...

The coiled-coil structure of the leucine zipper motif is not the only way that homodimers and heterodimers of transcription factors are formed. As we saw in Chapter 3 when discussing the RNA-binding protein ROP, the formation of a four-helix bundle structure is also a way to achieve dimerization, and the helix-loop-helix (HLH) family of transcription factors dimerize in this manner. In these proteins, the helix-loop-helix region is preceded by a sequence of basic amino acids that provide the DNA-binding site (Figure 10.23), and... 

The helix-turn-helix scaffold is designed to dimerize into a four-helix bundle. Modification of the peptide with the nicotinoyl functionality did not significantly perturb the peptide structure. CD spectroscopy showed no loss in helici-ty from the parent peptide and NMR spectroscopy confirmed successful incorporation of the nicotinoyl group as well as maintenance of crucial NOE connectivities. In particular, the presence of long-range NOE signals between the side chains of phenylalanine 38 and leucine 12 or isoleucine 9, which lie near the C- and AT-termini, respectively, demonstrate that the supersecondary structure of the motif has been conserved. 

Flg.1. In the amino acid sequence of KO-42 is encoded its fold and its function as it controls the formation of a hairpin helix-loop-helix motif that dimerizes to form a four-helix bundle. On the surface of the folded motif a reactive site is formed that catalyzes hydrolysis, transesterification and amidation reactions of reactive esters, whereas unfolded peptides are incapable of cooperative catalysis. In addition the values, and thus the reactivities, of the histidine residues are controlled by the fold. The pK of each His residue of KO-42 is shown in the figure and deviate by as much as 1.2 units from that of random coil peptides which is 6.4... 

Fig. 6. The high-resolution NMR structure of 2D, a 35-residue designed polypeptide that forms a four-helix bundle structure, showing the location of the aromatic residues in the core of the folded motif The cartoon illustrates the unexpected fold of a2D where the hairpin subunits dimerize in a interleaved mode. Reproduced with permission from J Am Chem Soc (1998) 120 1138. ( 1998 ACS)...

The 42-residue peptide KO-42 folds in solution into a hairpin helix-loop-helix motif that dimerizes to form a four-helix bundle. On the surface of the folded motif there are six histidines with assigned piC values in the range 5.2 to 7.2 (Fig. 1) and the second-order rate constant for the hydrolysis of mono-p-nitro-phenyl fumarate is 1140 times larger than that of the 4-methylimidazole-cataly-zed reaction at pH 4.1 and 290 K [13]. The reaction mechanism was found to be pH dependent as the kinetic solvent isotope effect was 2.0 at pH 4.7 and 1.0 at pH 6.1 and the pH dependence showed that the reaction rate depended on residues in their unprotonated form with piCj, values around 5. It was thus established that there are functional cooperative reactive sites that contain protonated and unprotonated His residues. 

As already discussed in Chapter 11, there are more than 10 000 protein structures known but only about 30 3D structure types. This might be traced to a limited number of possible stable polypeptide structures but most probably reflects the evolutionary history of the diversity of proteins. There are structural motifs which repeat themselves in a multitude of enzymes which are otherwise neither structurally nor functionally related, such as TIM barrel proteins, four-helix bundle proteins, Rossmann folds, or a/j3-folds of hydrolases (Figure 16.1). 

The overall three-dimensional structure of a protein is called the tertiary structure. The tertiary structure represents the spatial packing of secondary structures (Ofran and Rost, 2005). As for secondary structures, there are several different classes of tertiary structures. More advanced classification schemes take into account common topologies, motifs, or folds (Wishart, 2005). Common tertiary folds include the a/p-barrel, the four-helix bundle, and the Greek key (we will discuss protein folding further in Chapter 14). Any change to any part of the structure of a protein will have an impact on its biological activity (Thomas, 2003). 

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