Protein loop region

The secondary structure elements, formed in this way and held together by the hydrophobic core, provide a rigid and stable framework. They exhibit relatively little flexibility with respect to each other, and they are the best-defined parts of protein structures determined by both x-ray and NMR techniques. Functional groups of the protein are attached to this framework, either directly by their side chains or, more frequently, in loop regions that connect sequentially adjacent secondary structure elements. We will now have a closer look at these structural elements. 

Figure 2.8 Adjacent antiparallel P strands are joined by hairpin loops. Such loops are frequently short and do not have regular secondary structure. Nevertheless, many loop regions in different proteins have similar structures, (a) Histogram showing the frequency of hairpin loops of different lengths in 62 different proteins, (b) The two most frequently occurring two-residue hairpin loops Type I turn to the left and Type II turn to the right. Bonds within the hairpin loop are green, [(a) Adapted from B.L. Sibanda and J.M. Thornton, Nature 316 170-174, 1985.]...

Long loop regions are often flexible and can frequently adopt several different conformations, making them "invisible" in x-ray structure determinations and undetermined in NMR studies. Such loops are frequently involved in the function of the protein and can switch from an "open" conformation, which allows access to the active site, to a "closed" conformation, which shields reactive groups in the active site from water. 

The simplest motif with a specific function consists of two a helices joined by a loop region. Two such motifs, each with its own characteristic geometry and amino acid sequence requirements, have been observed as parts of many protein structures (Figure 2.12). 

Figure 2.12 Two a helices that are connected by a short loop region in a specific geometric arrangement constitute a helix-turn-helix motif. Two such motifs are shown the DNA-binding motif (a), which is further discussed in Chapter 8, and the calcium-binding motif (b), which is present in many proteins whose function is regulated by calcium.

Domains are formed by different combinations of secondary structure elements and motifs. The a helices and p strands of the motifs are adjacent to each other in the three-dimensional structure and connected by loop regions. Sequentially adjacent motifs, or motifs that are formed from consecutive regions of the primary structure of a polypeptide chain, are usually close together in the three-dimensional structure (Figure 2.20). Thus to a first approximation a polypeptide chain can be considered as a sequential arrangement of these simple motifs. The number of such combinations found in proteins is limited, and some combinations seem to be structurally favored. Thus similar domain structures frequently occur in different proteins with different functions and with completely different amino acid sequences. 

The proteins thus adapt to mutations of buried residues by changing their overall structure, which in the globins involves movements of entire a helices relative to each other. The structure of loop regions changes so that the movement of one a helix is not transmitted to the rest of the structure. Only movements that preserve the geometry of the heme pocket are accepted. Mutations that cause such structural shifts are tolerated because many different combinations of side chains can produce well-packed helix-helix interfaces of similar but not identical geometry and because the shifts are coupled so that the geometry of the active site is retained. 

The most frequent of the domain structures are the alpha/beta (a/P) domains, which consist of a central parallel or mixed P sheet surrounded by a helices. All the glycolytic enzymes are a/p structures as are many other enzymes as well as proteins that bind and transport metabolites. In a/p domains, binding crevices are formed by loop regions. These regions do not contribute to the structural stability of the fold but participate in binding and catalytic action. 

This is precisely where the catalytically essential zinc atom is found. This zinc atom is located precisely at this switch point, where it is firmly anchored to the protein by three side-chain ligands, His 69, Glu 72, and His 196 (Figure 4.20). The last residue of p strand 3 is residue 66, so the two zinc ligands His 69 and Glu 72 are at the beginning of the loop region that connects this p strand with its corresponding a helix. The last residue of p strand 5 is the third zinc ligand. His 196. 

In this structure the loop regions adjacent to the switch point do not provide a binding crevice for the substrate but instead accommodate the active-site zinc atom. The essential point here is that this zinc atom and the active site are in the predicted position outside the switch point for the four central parallel p strands, even though these p strands are only a small part of the total structure. This sort of arrangement, in which an active site formed from parallel p strands is flanked by antiparallel p strands, has been found in a number of other a/p proteins with mixed p sheets. 

The a/p-barrel structure is one of the largest and most regular of all domain structures, comprising about 250 amino acids. It has so far been found in more than 20 different proteins, with completely different amino acid sequences and different functions. They are all enzymes that are modeled on this common scaffold of eight parallel p strands surrounded by eight a helices. They all have their active sites in very similar positions, at the bottom of a funnel-shaped pocket created by the loops that connect the carboxy end of the p strands with the amino end of the a helices. The specific enzymatic activity is, in each case, determined by the lengths and amino acid sequences of these loop regions which do not contribute to the stability of the fold. 

A second example of up-and-down p sheets is the protein neuraminidase from influenza virus. Here the packing of the sheets is different from that in RBP. They do not form a simple barrel but instead six small sheets, each with four P strands, which are arranged like the blades of a six-bladed propeller. Loop regions between the p strands form the active site in the middle of one side of the propeller. Other similar structures are known with different numbers of the same motif arranged like propellers with different numbers of blades such as the G-proteins discussed in Chapter 13. 

A more complex p helix is present in pectate lyase and the bacteriophage P22 tailspike protein. In these p helices each turn of the helix contains three short p strands, each with three to five residues, connected by loop regions. The p helix therefore comprises three parallel p sheets roughly arranged as the three sides of a prism. However, the cross-section of the p helix is not quite triangular because of the arrangement of the p sheets. Two of the sheets are... 

The number of possible ways to form antiparallel p structures is very large. The number of topologies actually observed is small, and most p structures fall into these three major groups of barrel structures. The last two groups—the Greek key and jelly roll barrels—include proteins of quite diverse function, where functional variability is achieved by differences in the loop regions that connect the p strands that build up the common core region. 

The second protein in the membrane of influenza vims, neuraminidase, does not belong to any of these three groups of barrel structures. Instead, it forms a propeller-like structure of 24 p strands, arranged in six similar motifs that form the six blades of the propeller. Each motif is a p sheet of 4 up-and-down-connected p strands. The enzyme active site is formed by loop regions on one side of the propeller. 

The elegant genetic studies by the group of Charles Yanofsky at Stanford University, conducted before the crystal structure was known, confirm this mechanism. The side chain of Ala 77, which is in the loop region of the helix-turn-helix motif, faces the cavity where tryptophan binds. When this side chain is replaced by the bulkier side chain of Val, the mutant repressor does not require tryptophan to be able to bind specifically to the operator DNA. The presence of a bulkier valine side chain at position 77 maintains the heads in an active conformation even in the absence of bound tryptophan. The crystal structure of this mutant repressor, in the absence of tryptophan, is basically the same as that of the wild-type repressor with tryptophan. This is an excellent example of how ligand-induced conformational changes can be mimicked by amino acid substitutions in the protein. 

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