The Quaternary Structure Of A Protein Biology Essay

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Proteins are the most biologically abundant and functionally diverse molecules found in cells: The catalytic nature of many proteins help create and regulate the metabolic pathways that supply our cells with the required energy and nutrients, while others function as transporters, structural building blocks, transcription factors, immunological response mechanisms, motility apparatus, or communication systems. Regardless of whatever the highly specific function of a protein may be, it always requires a highly specific structure. Thus the interactions of these proteins, between both their subunits and their substrates, on a molecular level are due to the preceding assembly of a concise quaternary structure.

The quaternary structure is defined as the association of several polypeptide chains, each with their own domain regions, to form a multi-subunit molecule. There are many functional benefits to this structural formation:

  • Subunit association helps combine domains in ways that bring about active site complexes (i.e.: those of glutamine synthetase, shown later) or allow related reactions to take place in quick succession.
  • The combining of subunits decreases the surface-volume ratio which is highly beneficial to protein stability since there are less destabilising environmental interactions (destabilising in aqueous solution) and more thermodynamically favourable subunit-subunit interactions. The presence of hydrophobic regions in monomers will induce a very rapid conformation to a very stable complex.
  • Quaternary structure formation can, in some cases, increase the affinity of substrate binding. As a ligand binds to a monomer, the affinity of the other monomers for their respective ligands increases. This cooperative binding is seen in many molecules, such as haemoglobin, and is a frequent mechanism for enzyme regulation [1].

The two typical categories of quaternary structures encompass those with subunits which are structurally diverse, and those which contain multiple copies of the same subunits. In the former group, the non-identical subunits lead to an irregular geometric pattern through singular interactions between one another. Many proteins possess dissimilar yet interacting domains which combine their individual functions to give an overall enzymatic effect [2]. Phosphorylase kinases (PHK), for example, house an ADP binding domain in their β subunits, calcium ion binding in the calmodulin (δ) subunits, and a kinase domain in the γ subunits [3]. This quaternary structure is useful in illustrating the hybridisation of many different domains; however, the individual domains themselves can be used by many other proteins while often remaining highly conserved in their structure. A β subunit, for example, which is associated with purine binding, is found in both mitochondrial [4], and E. coli [5] ATP synthase as well as PHK since the binding of ADP is a shared function of the two enzymes.

The alternative category for quaternary structure, commonly seen as the structural formations of viral capsids, involves repeating interactions between recurring subunits. Such structures can take on either a linear or, more frequently, a helical formation (The Tobacco Mosaic Virus nucleocapsid, for example, is typically comprised of 2,130 identical subunits) that can be easily extended by the simple addition of further subunit repeats. These structures produce a geometrically regular structure. In the case of TMV nucleocapsid, the regular helical structure surrounds the viral RNA functioning as protection from the environment. The 60 subunit capside used by bacteriophage λ expresses icosahedral geometry and is another good example for repeating subunit structures [6].

The functioning domains of each subunit in a quaternary structure can remain independent of one another or work in regulatory conjunction. The term domain entails part or all of a polypeptide sequence which can fold into a three dimensional structure that carries a function, for example; proteolysis. These domain regions are comprised of several supersecondary elements which give rise to the smaller details of a protein's structure such as α helixes, β sheets, and turns. These elements, or motifs, can carry functions of their own such as DNA or ligand binding. Thus their individual structure, and their position in the greater tertiary and quaternary structures, is highly important in inferring the intended function of a protein.

Glutamine Synthetase


Glutamine Synthetase (GS) is catalytically essential for the biosynthesis of glutamine from glutamate and ammonium in a two step mechanism, in which the intermediate is activated γ-glutamyl phosphate (Figure 1). The binding of two divalent cations (Me+) helps to transfer the phosphate group (n2 binding site) of ATP to the carboxyl group of glutamate, as well as stabilise the active enzyme (n1 binding site). The phosphate group is subsequently replaced for an amine by the ammonium ion.

The structure of GS is such that the substrates, including the intermediate, are tightly bound to the enzyme complex, thus allowing for reversible reactions. GS is able to perform a transferase activity which is partially the reverse of glutamine synthesis. Hydroxylamine and glutamine can react to produce γ-glutamylhydroxamate and NH4+ (ATP[1] and the cations must still be present). The transferase activity is derived from the hydroxylamine replacing the phosphate (or arsenate) group of the formed intermediate γ-glutamyl arsenate [7].

The hydroxyl group of a water molecule can induce glutamine hydrolysis activity in GS, as the intermediate arsenate group is replaced by –OH thus reforming glutamate. These three reactions all relate to structure since their reaction mechanisms all imply that the same binding site can conform to fit ammonium ions (biosynthesis), hydroxylamine (transferase), and deprotonated water (hydrolysis). The other binding sites are important for important for the other reactants which are shared across these processes [8].

All these reactions infer a high level of cooperativity between GS subunits since all substrates must be present in the active site before any catalysed exchanges can take place. The order of substrate binding is also crucial and if incorrect will result in no formation of γ-glutamyl phosphate [9, 10].

The relation to structure:

The bacterial glutamine synthetase enzyme is built up of two hexameric rings which link together through mainly hydrophobic interactions to create a dodecamer protein, consisting of twelve active sites [11]. Each monomer forms part of an active site with its adjacent subunit along the 6-fold axis, thus the active sites are formed between subunits. As seen by the preceding description of catalytic function, the active sites need to bind ATP, two divalent cations, glutamate, and ammonium. The domain structure that allows this is shaped like an hourglass or ‘bifunnel', in which the ATP binds to the top near the N-terminal helix which is exposed to the surrounding solvent, and glutamate binds at the opposite end to ensure correct orientation during reaction. Binding sites n1 and n2 fix magnesium or manganese ions to the centre of the domain in a heavily antiparallel β sheet region. A monovalent binding site which is believed to be the location for ammonium cation binding [7] is present adjacent to the glutamate binding site; ligand residues being glu212, tyr179, asp50', ser53'[2].

Figure 2 below shows the rough positions of substrates in the active site and throughout glutamine synthesis. Figure 3 provides a detailed ribbon structure indicating the locations of the two cations as reference points. Figure 3 shows eight β sheets forming a partial barrel structure at the active site core. The C-terminal helix sticks out of the bottom and inserts itself into a hydrophobic hole in the opposite subunit of the other hexamer. This region is called the ‘helical thong' and provides the quaternary structure with greater adhesion. These two rings are further held in place by β loops provided by opposing monomers on each ring which line up in an antiparallel manner in the central cavity of the proteins. These six β formations make up the central flexible loop region and become the potential cleaving site for proteolysis [12].

The quaternary structure of GS is evidently crucial for function, since it is the combining of subunits which form the active sites. Nucleotide binding on the N-terminus of one subunit causes conformational alterations of residues in the neighbouring subunit (asp50' and asp64') resulting in increased stability and affinity for ammonium binding.

The dodecameric structure of GS is only applicable to prokaryotic life, with small variation throughout (S. Typhimurium GS is 620kDa whereas M. Tuberculosis GS is 640kDa) [8]. The structure of GS in eukaryotesis highly altered (less than 20% sequence similarity); consisting of three pentamer ring structures[3] [13]. Hence the structure is comprised of 15 subunits, rather than 12, however only 10 active sites are present since one of the pentamers is non-catalytic. The other two pentamers are connected by specific hydrogen bonds although no ‘helical thong' is present.

The general hourglass shape of an active site in GS is shown. The grey circles signify the n1 and n2 sites. a) The third phosphate of ATP binds next to the n2 ion. Asp50' moves the subsequent site of ammonium binding. b) The carboxly group of glutamate orientates next to the n1 ion. c The γ-glutamyl phosphate is formed. d&e) Ammonium binds at a negatively charged pocket and becomes deprotonated to ammonia by the Asp50'. f) phosphate on the carboxyl is substituted by the ammonia. g&h) the glu327 flap neutralises the ammonia group and subsequently releases the newly formed glutamine product [8].

The subunits within the pentamers are made up of a β-grasp domain at the N-terminus, which is involved in subunit-subunit interactions and active site formations, and a catalytic domain at the C-terminus. There is high conservation between prokaryotic and eukaryotic GS around the cation binding sites; however, there is no residue similarity at the nucleotide binding site. Figure 4 shows how two proteins with very different structural formations can give the same functional output, despite major variations in enzymatic and inhibitory details.

Influenza Virus Haemagglutinin


Haemagglutinin (HA) is located on the surface of the Influenza virus and is debatably the more important of two membrane fusogen proteins, the other being neuraminidase. It is responsible for virus-host attachment and envelope-membrane fusion, making it vital for virus infection. HA is designed to bind to the terminal sialic acid residue of cellular glycoproteins which act as the receptors. This attachment induces invagination of the cell membrane and the virus is taken in by endocytosis, (fig 5.1). The proton pumps on the newly synthesised vesicle generate and maintain a low pH environment (fig 5.2). This causes a conformational change to the HA structure which brings the viral envelope closer to the membrane and exposes its fusion peptides (fig 5.3 & 5.4). Upon membrane fusion the virus releases its RNA into the cytoplasm which then moves through the nuclear pores (fig 5.5).

Relation to Structure:

HA is a homotrimeric protein (~210kDa) made up from glycosylated subunits comprised of a filament region (HA2 & HA1) which extends out into a globular region (HA1) at the end. During production of HA, the precursor for the monomer is one long chain which, during translocation, becomes connected to the outside of the membrane by a signal N-terminal sequence. This precursor undergoes proteolytic cleavage at the 329 residue thus producing the polypeptides HA1 (316 residues) and HA2 (210 residues), which are linked by disulphide bridges to one another. Glycosylation of residues 11, 23, 163, and 277 on HA1, and just 156 on HA2 is made through N-acetylglucosamine-asparagine linkages, with additional sugars attached [16]. These carbohydrates make up ~19% by weight of the overall glycoprotein [17].

The main feature of HA1, the globular region and part of the stem region, is the presence of an obtuse jelly roll structure (resides 116-261). Eight β sheets partially align in an antiparallel manner to form a disfigured barrel shape. The top of this motif is the site for receptor binding with sialic acid (Figure 6); the central amino acids of this small pocket are highly conserved but surrounded by variable residues. The fact that sialic acid covers the entire pocket means that antibodies which can bind to the variable region will prevent virus infection due to receptor binding inhibition [18]. A secondary binding site exists between the interface of two HA1 and one HA2 domains, which is able to bring about close contact, by binding to a sialic acid derivative, but must dissociate in order for the virus to orientate itself for membrane fusion. This site is at least four times weaker than the primary binding site and at current has no evidential physiological significance [19, 20].

The sequences either side of the jelly roll structure feed out of and back into the stem region of the HA monomer, in an antiparallel fashion. The stem region (HA2) is designed so that the receptor binding site can protrude out from the membrane as far as possible. Its main motif feature is a long α helix hairpin loop which stretches out 76Å. The antiparallel regions of HA1 are surrounded either side by β sheets of HA2 which help bond the two polypeptides together.

The N-terminus of HA2 (~ 20 residues) is the most conserved region of the protein and constitutes the fusion peptide mentioned earlier. Thus a conformational change must occur in the quaternary structure for this region, which is well embedded in the surrounding polypeptide structure due to its hydrophobic nature[4], to penetrate the cell surface [21]. This conformational change can be induced by high temperatures, but more importantly by low pH environments such as that of the endosome, and results in the movement of the fusion peptide by more than 100Å (>10nm). The three α helixes, from the HA2 monomers, intertwine to form a spiral which, upon environmental alteration, unfold at the middle and invert 180o [22, 23] forcing the C-terminal of the helixes backwards and bringing the N-terminal round to place near the host membrane [24]. The disulphide bridge from residue 14 on HA1 becomes reduced (due to the increasingly acidic environment) causing dissociation from HA2 around the globular head indicating movement to allow space for fusion peptide entry [25]. The helix loop region in the HA2 subunit acts as a hinge to allow spreading out of the three peptides over the host surface. Virus-cell fusion and genetic transfer consequently takes place.

This low-pH induced conformation has been found experimentally to be irreversible and have an improved thermostable nature [26]. Therefore, the neutral-pH conformation of HA must be metastable. This accounts for the ability of HA to perform such an elaborate unfolding/refolding mechanism; since the metastable state, confined within the quaternary structure, will naturally change formation under low pH to become thermodynamically stable. Few proteins are able to maintain a metastable conformation. Haemagglutinin is a rare example of the thermodynamic and kinetic influence the quaternary structure can have on a protein, rather than simply bringing different functional domains into closer proximity.

Discussion and Conclusion:

GS and HA are two examples of proteins with intricate quaternary structures orientated in a highly specific manner which deduces their function. In the case of GS, the sites of catalysis are generated from the combining of two subunits and so without bonding between polypeptides there could be no enzyme. The final structure of HA is designed to allow receptor binding and then undergo conformational change to bring the virus and host membranes into close contact. Thus, its structure is highly developed for its role in influenza virus attachment and entry.

These two examples demonstrate the linkage between quaternary structure and function. However, many proteins are made from only one polypeptide chain and consequently have no quaternary structure. None the less, they are able to function independently and make up many vital housekeeping proteins. DNA polymerase I, for example, is one long polypeptide chain which contains three separate domains for each of its three functions: 5'-3' exonuclease activity (residues 1 to 324), 3'-5' exonuclease activity (325 to 518), and polymerase activity (519 to 928)[5]. Therefore, quaternary structures, especially in the cases of enzymes, are not always necessary. What determines whether a protein will have a final tertiary structure or quaternary structure may be related to evolutionary divergences, or genetic conservation to maintain a smaller genome and prevent the same domains being coded repeatedly for different proteins.

It is much more obvious why structural proteins have quaternary structures since repeat subunits are aligning in regular patterns, just like building blocks, to create a large geometric complex. If this were done from a single polypeptide it would be far too big and unstable to effectively function as surrounding protection for a viral genome.

The fact that quaternary structures can be broken down into subunits, unlike tertiary structures, could have potential for protein engineering. Knowledge of the different domains and how they relate to each other in a complex could allow insight into the development of synthetic proteins; spliced together from the domains of several subunits. Alternatively, the designing of a monomer that can form a repeat structure could create new methods for vector uptake by placing them inside these artificial configurations.

The function of a protein is, in all cases, derived from the assumed structure that it acquires. All stages of protein structure are related to one another and thus related to function. However, the quaternary structure, as has been described using examples, stands out on a level of increased complexity in structural formation and has been shown to generate functions that are otherwise impossible without the conjugation of polypeptides.


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[1] A nucleotide arsenate can be used instead of ATP. ADP also functions effectively.

[2] The ‘next to a residue indicates that it is on the neighbouring subunit. This helps show how the quaternary structure forms the active sites.

[3] The eukaryote used in experimentation by Krajewski [13] is Canis Familiaris.

[4] The hydrophobic nature allows this fusion peptide to easily fuse with the host cells membrane

[5] The polymerase and 3'-5' exonuclease domains constitute the Klenow Fragment.