The tailspike protein (P22TSP) of

Enterobacteria phage P22 Salmonella virus P22 is a bacterial virus (bacteriophage) that infects ''Salmonella typhimurium''. Like many phages, it has been used in molecular biology to induce mutations in cultured bacteria and to introduce foreign genetic material. P22 ha ...

mediates the recognition and adhesion between the

bacteriophage A bacteriophage (), also known informally as a phage (), is a virus that infects and replicates within bacteria. The term is derived . Bacteriophages are composed of proteins that Capsid, encapsulate a DNA or RNA genome, and may have structu ...

and the surface of ''

Salmonella enterica ''Salmonella enterica'' (formerly ''Salmonella choleraesuis'') is a rod-shaped, flagellate, facultative anaerobic, Gram-negative bacterium and a species of the genus ''Salmonella''. It is divided into six subspecies, arizonae (IIIa), diarizonae ...

'' cells. It is anchored within the viral coat and recognizes the O-antigen portion of the

lipopolysaccharide Lipopolysaccharide (LPS), now more commonly known as endotoxin, is a collective term for components of the outermost membrane of the cell envelope of gram-negative bacteria, such as '' E. coli'' and ''Salmonella'' with a common structural archit ...

(LPS) on the outer-membrane of

Gram-negative bacteria Gram-negative bacteria are bacteria that, unlike gram-positive bacteria, do not retain the Crystal violet, crystal violet stain used in the Gram staining method of bacterial differentiation. Their defining characteristic is that their cell envelo ...

. It possesses endoglycanase activity, serving to shorten the length of the O-antigen during infection.

History

The initial interest in tailspike proteins was in the study of the effect the mutations on

protein folding Protein folding is the physical process by which a protein, after Protein biosynthesis, synthesis by a ribosome as a linear chain of Amino acid, amino acids, changes from an unstable random coil into a more ordered protein tertiary structure, t ...

. Some mutations affect the folding efficiency of the protein but have no effect on the final

native Native may refer to: People * '' Jus sanguinis'', nationality by blood * '' Jus soli'', nationality by location of birth * Indigenous peoples, peoples with a set of specific rights based on their historical ties to a particular territory ** Nat ...

structure. Other mutations have been identified that lead to a temperature sensitive phenotype. Reconstitution experiments have demonstrated that the

in vitro ''In vitro'' (meaning ''in glass'', or ''in the glass'') Research, studies are performed with Cell (biology), cells or biological molecules outside their normal biological context. Colloquially called "test-tube experiments", these studies in ...

folding Fold, folding or foldable may refer to: Arts, entertainment, and media * ''Fold'' (album), the debut release by Australian rock band Epicure * Fold (poker), in the game of poker, to discard one's hand and forfeit interest in the current pot *Abov ...

process closely mirrors the

in vivo Studies that are ''in vivo'' (Latin for "within the living"; often not italicized in English) are those in which the effects of various biological entities are tested on whole, living organisms or cells, usually animals, including humans, an ...

pathway. It has been further been demonstrated that folding yields in vitro decrease strongly with increasing temperature.

Function

O-antigen binding

P22TSP recognizes the O-antigen polysaccharide of LPS serotypes A, B, or D1. The serotypes correspond to species ''S. Typhimurium'', ''S. Enteritidis'', and ''S. Paratyphi A''. These

carbohydrate A carbohydrate () is a biomolecule composed of carbon (C), hydrogen (H), and oxygen (O) atoms. The typical hydrogen-to-oxygen atomic ratio is 2:1, analogous to that of water, and is represented by the empirical formula (where ''m'' and ''n'' ...

s share the same main chain trisaccharide repeating unit alpha-D-

mannose Mannose is a sugar with the formula , which sometimes is abbreviated Man. It is one of the monomers of the aldohexose series of carbohydrates. It is a C-2 epimer of glucose. Mannose is important in human metabolism, especially in the glycosylatio ...

-(1—4)-alpha-L-

rhamnose Rhamnose (Rha, Rham) is a naturally occurring deoxy sugar. It can be classified as either a methyl- pentose or a 6-deoxy- hexose. Rhamnose predominantly occurs in nature in its L-form as L-rhamnose (6-deoxy-L- mannose). This is unusual, since mo ...

-(1—3)-alpha-D-

galactose Galactose (, ''wikt:galacto-, galacto-'' + ''wikt:-ose#Suffix 2, -ose'', ), sometimes abbreviated Gal, is a monosaccharide sugar that is about as sweetness, sweet as glucose, and about 65% as sweet as sucrose. It is an aldohexose and a C-4 epime ...

-(1—2), but each have a different 2,6-dideoxyhexose substituent at C-3 of the

In vivo Studies that are ''in vivo'' (Latin for "within the living"; often not italicized in English) are those in which the effects of various biological entities are tested on whole, living organisms or cells, usually animals, including humans, an ...

, P22TSP binds as a homotrimer and one

phage A bacteriophage (), also known informally as a phage (), is a virus that infects and replicates within bacteria. The term is derived . Bacteriophages are composed of proteins that encapsulate a DNA or RNA genome, and may have structures tha ...

particle can carry up to 6 tailspikes. P22TSP can bind multivalently, leading to an essentially irreversible attachment. It was shown that a minimum of two repeating units or an octasaccharide is required for binding. The TSP is also capable of binding longer fragments with similar affinity.

Endoglycosidase activity

P22TSP has endorhamnosidase activity and cleaves the

glycosidic bond A glycosidic bond or glycosidic linkage is a type of ether bond that joins a carbohydrate (sugar) molecule to another group, which may or may not be another carbohydrate. A glycosidic bond is formed between the hemiacetal or hemiketal group o ...

of the rhamnose group, producing an octasaccharide product. Two

aspartic acid Aspartic acid (symbol Asp or D; the ionic form is known as aspartate), is an α-amino acid that is used in the biosynthesis of proteins. The L-isomer of aspartic acid is one of the 22 proteinogenic amino acids, i.e., the building blocks of protei ...

s and one

glutamic acid Glutamic acid (symbol Glu or E; known as glutamate in its anionic form) is an α- amino acid that is used by almost all living beings in the biosynthesis of proteins. It is a non-essential nutrient for humans, meaning that the human body can ...

in the active site have been strongly linked to

enzymatic activity Enzyme assays are laboratory methods for measuring enzymatic activity. They are vital for the study of enzyme kinetics and enzyme inhibition. Enzyme units The quantity or concentration of an enzyme can be expressed in molar amounts, as with any ...

. Different biological functions for this cleavage have been proposed. Cleavage could facilitate access to the

membrane A membrane is a selective barrier; it allows some things to pass through but stops others. Such things may be molecules, ions, or other small particles. Membranes can be generally classified into synthetic membranes and biological membranes. Bi ...

. or allow the

to find the optimal position for

infection An infection is the invasion of tissue (biology), tissues by pathogens, their multiplication, and the reaction of host (biology), host tissues to the infectious agent and the toxins they produce. An infectious disease, also known as a transmis ...

Role in DNA injection

It has been demonstrated that cleavage of the O-antigen is necessary for

DNA Deoxyribonucleic acid (; DNA) is a polymer composed of two polynucleotide chains that coil around each other to form a double helix. The polymer carries genetic instructions for the development, functioning, growth and reproduction of al ...

ejection by the

. It has been proposed that P22TSP binding positions the

to inject its

Structure

P22TSP is a homotrimeric structural protein consisting of 666

amino acid Amino acids are organic compounds that contain both amino and carboxylic acid functional groups. Although over 500 amino acids exist in nature, by far the most important are the 22 α-amino acids incorporated into proteins. Only these 22 a ...

s. It is noncovalently bound to the neck of the viral

capsid A capsid is the protein shell of a virus, enclosing its genetic material. It consists of several oligomeric (repeating) structural subunits made of protein called protomers. The observable 3-dimensional morphological subunits, which may or m ...

. It has been crystallized in

space group In mathematics, physics and chemistry, a space group is the symmetry group of a repeating pattern in space, usually in three dimensions. The elements of a space group (its symmetry operations) are the rigid transformations of the pattern that ...

P213 and has one

monomer A monomer ( ; ''mono-'', "one" + '' -mer'', "part") is a molecule that can react together with other monomer molecules to form a larger polymer chain or two- or three-dimensional network in a process called polymerization. Classification Chemis ...

in the asymmetric unit. The secondary structure of P22TSP is dominated by a parallel

Beta helix A beta helix is a tandem protein repeat structure formed by the association of parallel beta sheet in a helical pattern with either two or three faces. The beta helix is a type of solenoid protein domain. The structure is stabilized by inter-stra ...

comprising 13 complete turns. This structure is further characterized as a beta-solenoid domain. P22TSP is compOsed of two domains, each with distinct function. An

N-terminal The N-terminus (also known as the amino-terminus, NH2-terminus, N-terminal end or amine-terminus) is the start of a protein or polypeptide, referring to the free amine group (-NH2) located at the end of a polypeptide. Within a peptide, the amin ...

domains serves to bind to the

particle and a

C-terminal The C-terminus (also known as the carboxyl-terminus, carboxy-terminus, C-terminal tail, carboxy tail, C-terminal end, or COOH-terminus) is the end of an amino acid chain (protein or polypeptide), terminated by a free carboxyl group (-COOH). When t ...

domains that interacts with the ''

Salmonella ''Salmonella'' is a genus of bacillus (shape), rod-shaped, (bacillus) Gram-negative bacteria of the family Enterobacteriaceae. The two known species of ''Salmonella'' are ''Salmonella enterica'' and ''Salmonella bongori''. ''S. enterica'' ...

'' surface. These two domains are connected by a flexible linker. The

binding site In biochemistry and molecular biology, a binding site is a region on a macromolecule such as a protein that binds to another molecule with specificity. The binding partner of the macromolecule is often referred to as a ligand. Ligands may includ ...

of P22TSP is located in the central part of the

. A deep cleft is formed by a 60- residue insertion on one side along with three smaller 5-25 residue insertions on the other.

Homologous proteins

Several functional homologues of P22TSP has been identified belonging to the

s HK620 and Sf6. Both of these tailspike

protein Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residue (biochemistry), residues. Proteins perform a vast array of functions within organisms, including Enzyme catalysis, catalysing metab ...

s also contain right-handed parallel beta-helices and share similar O-antigen binding and cleavage to P22TSP. These proteins share 70%

sequence identity In bioinformatics, a sequence alignment is a way of arranging the sequences of DNA, RNA, or protein to identify regions of similarity that may be a consequence of functional, structural, or evolutionary relationships between the sequences. Align ...

in their

domains, but no sequence similarities have been found in the

domains.

Translational applications

Carbohydrate binding scaffolds

P22TSP has also been studied due to its high kinetic stability. As it exists and functions in the

extracellular This glossary of biology terms is a list of definitions of fundamental terms and concepts used in biology, the study of life and of living organisms. It is intended as introductory material for novices; for more specific and technical definitions ...

environment, it must endure harsh conditions such as highly variable temperatures or high concentrations of protein degrading

enzyme An enzyme () is a protein that acts as a biological catalyst by accelerating chemical reactions. The molecules upon which enzymes may act are called substrate (chemistry), substrates, and the enzyme converts the substrates into different mol ...

s. The kinetic stability of P22TSP derives from its compact beta-solenoid architecture. It was shown that like other viral fibrous proteins, P22 tailspike protein possesses a high stability against denaturation. This makes P22TSP a promising candidate for use as thermostable scaffold capable of being tailor-made to recognize heteropolymers.

Therapy against ''Salmonella'' infection

Tailspike proteins have also shown potential for more translational applications such as fighting

bacteria Bacteria (; : bacterium) are ubiquitous, mostly free-living organisms often consisting of one Cell (biology), biological cell. They constitute a large domain (biology), domain of Prokaryote, prokaryotic microorganisms. Typically a few micr ...

s. A study has demonstrated that orally administered P22TSP markedly reduced ''

'' colonization in a group of chickens. They suggest that the endorhamnosidase activity of the free tailspike molecule serves to modify O-antigen, compromising the LPS structure and thereby preventing the binding of a phage P22-attached tailspike protein. The authors suggest that this has the potential to be a novel therapy meant to fight

References

{{Reflist, 33em Proteins