STRUCTURAL BIOLOGY Alpha Domains By Assist. Prof. Dr. Betul Akcesme

Classification of Domains Protein domains can be classified according to their secondary structural elements Five broad classes Alpha domains are comprised entirely of alpha helices. Beta domains contain only beta sheet. Alpha/beta domains contain beta strands with connecting helical segments. Alpha+beta domains contain separate beta sheet and helical regions. Cross-linked domains have little, if any, secondary structure but are stabilized by several disulfide bridges or metal ions.

ALPHA DOMAIN…

Packing together, they are more stable While isolated α helices often occur in proteins, they are only marginally stable. They are stabilized by being packed together through hydrophobic side chains. We will worry about membrane proteins later.

1)Coiled coil In 1953, Francis Crick showed that the side chain interactions are maximized if the two alpha helices are wound around each other in a “coiled coil” arrangement rather than being straight rods. Such structures are the basis of some fibrous proteins, and others. For fibers, sometimes many hundreds of amino acids to make long flexible dimmer. Shorter coiled-coils are also used elsewhere. Left handed supercoil from two right-handed helices

The heptad repeat Number of residues per turn reduced from 3.6 to 3.5. The pattern of side chain interaction repeats every 7 residues, after 2 turns. Such sequence is repetitive with period 7. One heptad repeat is labeled a-g. Residue d is hydrophobic: often a leucine. Residue a is also hydrophobic The side chains of e and g (often charged a.a.) form ionic interactions between 2 helices Transcription Factor GCN4

Heptad structure and prediction? ‘d’ residue packs against each other, ‘a’ residue packs against each other. Heptad repeats provide strong indications of alpha helical coiled coil structures. They are found in many proteins of diverse functions. Fibrinogen, collectins, muscle protein myosin, DNA- RNA binding proteins and so on.. predicting such structures? This not only helps to predict secondary but also double helices, helping 3-D prediction.

Holding up two helices: Knobs in Holes model ‘a’ packs with ‘a’, and ‘d’ with ‘d’ (2 levels squashed view) The e-g salt bridges stabilize coiled coil structures. Just one side of helices, From e’s to g’s.

Crick’s Knobs and hole model Alpha helices pack against each other according to knobs and hole model: Side chain in the hydrophobic region of one alpha helix can contact 4 side chains from the other helix. The side chain of the residue at position d is directed into a hole at the surface of the other helix, surrounded by 1 ‘d’, 2 ‘a’’s, 1 ‘e’, with numbers n, n-3, n+4, n+1, resp. Two ‘d’s face each other (usually leucine, isoleucine) knobs holes Superimpose Two parallel helices

Example for Knobs in Holes(KIH)  Views of a classical coiled-coil dimer. (A and B) Orthogonal views of the structure. with the backbone represented by ribbons, side chains that make up the KIH interactions as sticks and the heptad positions a–g coloured red to violet. In this way, the hydrophobic core can be easily followed in red and green. (C) A single KIH interaction between a ‘knob’ from one helix (shown in green on the rightmost helix) and a ‘hole’ formed by four residues on the other (left helix). Images were created using PyMOL (8) and the PDB entry 2ZTA (9). KNOB 1 2 3 HOLE 4 Example for Knobs in Holes(KIH)

2)Four helix bundle Two alpha helices are not enough to build a domain. The simplest and most frequent alpha- helical domain is 4 alpha helix bundle. Green = hydrophobic residues, packed inside hydrophobic core; red = hydrophilic residues. 20o This appears in many proteins for oxygen transport, electron carriers, storing iron atoms, coat protein for some virus.

4 helix bundle Usually in 4 helix bundle adjacent helices are antiparallel Two pairs of parallel helices, joined in antiparallel fasion. Two such 4 helix bundle are packed according to the “ridges and grooves” model. (As in pictures) Ridges of one alpha helix will fit into grooves of an adjacent helix Cytochrome b562 Human growth hormone

A pair of coiled coils packed with knobs in holes model Rop protein has two subunits. Each subunit is an antiparallel coiled-coil in which the hydrophobic side chains are packed against each other according to the knobs and holes model. Then two such subunits are packed according to the “ridges and grooves” model. Rop protein

Large and complex ones Several enzymes have long polypeptide chain of 300-400 a.a. arranged in over 20 alpha helices packed together in a complex pattern to form a globular domain. Bacterial muramidase: 618 aa. 450 aa at the N-terminal side form a alpha-helical domain 27 alpha helices 2 layered ring Diameter 30A Right handed super twist

3) The globin fold Contains iron Globin fold: 8 alpha helices: A-H. has been found in many related proteins: myoglobin, hemogobin, light capturing assemblies in algae, … Very different from 4 helix bundle, The 8 helices are connected by short loop regions, forming a pocket for active site (this site for myoglobin and hemoglobin binds a heme group). Length varies from 7 (C) to 28 (H) in myoglobin. Adjacent helices are not sequentially adjacent (except for G and H which form antiparallel pair). +90 and +50 angles with respect to each other Not from smaller motifs. Globin fold: 8 alpha helices: A-H. The white is heme group.

Globins from dif organisms determined and 99% to 16% sequence homology They share the essential features of globin fold very diverge aa sequence but similar 3D structure BUT HOW?

Evolution: what affects protein fold? People studied 9 globin structures, trying to figure our what positions are important for protein to be folded that way. Examined in detail residues at structurally equivalent positions involved in helix heme contacts and in packing the alpha helices against each other They found 59 positions – 31 buried in the interior of protein 28 positions in contact with heme group. They are the principle determinants of both function and 3D structure of globin family They found that there is a striking preferential conservation of the hydrophobic character of the amino acids at the buried positions. On the other hand, at the exposed positions, it actually does not matter whether we have mutations from hydrophilic to hydrophobic or vice versa, with one exception: the amino acid 6 in the beta chain in myoglobin (sickle cell disease)

A change in residue 6 of the beta chain turn from GLUTAMIC ACID TO VALIN

Sickle-cell hemoglobin E6V mutation

Hydrophobic pocket of deoxyganated form of

Cartoon illustration of how AS heterozygotes are relatively protected from severe P falciparum malaria. The upper part of the cartoon is a schematic diagram of what happens in red cells in a normal (Hb AA) person with malaria: after invasion of a red cell by a merozoite, this becomes a ring form, and this starts multiplying (schizogony) ; when a schizont is mature the infected red cell essentially bursts and releases new merozoites, each one of which can invade a new red cell. The lower part of the cartoon is a schematic diagram of what happens in red cells in an AS heterozygote with malaria: the red cell, which appears normal at the time of invasion, once infected undergoes sickling (probably as a result of deoxygenation and lowering pH caused by the parasite), and thus it falls easy prey to macrophages in the spleen, in other organs and even in the peripheral blood42. Phagocytosis of a parasitized red cells clearly interrupts the schizogonic cycle and thus the parasitaemia can be kept under control.