Linker

Linkers or spacers are short amino acid sequences created by nature to separate multiple domains in a single protein. Natural linkers thus act as a kind of spacer between the domains of multidomain proteins and serve to prevent unwanted interactions between the individual domains.The length of linkers varies from 2 to 31 amino acids. Linkers are optimized for each condition in such a way that they do not impose any restrictions on the conformation or interactions of the linked partners.

Rigid linkers: Most linkers are rigid spacers. The majority of interdomain linker sets consist of Pro, Arg, Phe, Thr, Glu, and Gln residues. Proline is common to many naturally occurring interdomain linkers. Structural studies suggest that proline-rich sequences form relatively rigid extended structures to prevent unfavorable inter-domain interactions. Independent analysis showed that Thr, Ser, Gly, and Ala are also preferred residues in natural linkers.

Flexible linkers: Gly-rich linkers, on the other hand, are flexible rather than rigid. Gly-rich linkers have been shown to be useful for unstable interactions, especially when the interaction is weak and transient. The linkers establish a covalent bond between the proteins. This results in the formation of a stable protein-protein complex. Gly-rich linkers are also used to form stable covalently linked dimers and connect two independent domains forming a ligand binding site or recognition sequence. In addition, flexible Gly-rich regions have been observed as natural linkers in proteins, creating loops that connect domains in multidomain proteins.

With the advent of recombinant DNA technology, it became possible to link two interacting partners by introducing artificial linkers. By linking binding partners with an artificial linker, the proximity between the interacting partners is increased and the natural interaction is maintained.

Meanwhile, it has been revealed that naturally occurring Gly-rich linkers have functional roles in protein in addition to linking domains. For example, the transcription factor PAX6 consists of two DNA-binding domains, a paired domain (PD) and a homeodomain (HD), connected by a Gly-rich linker. Crystal structure analysis of the human PAX6-PD-DNA complex revealed that the extended linker makes contacts with DNA in the minor groove.

In the transmembrane glycoproteins (TMs) of retroviruses, linkers also play an important role by mediating membrane fusion through an N-terminal fusion peptide. The fusion peptide is connected to the central coiled-coil core via Gly-rich linkers. The length and amino acid composition of the linkers are conserved in many retroviruses and in HA2 of influenza viruses.

1. Reddy Chichili VP et al (2013) Linkers in the structural biology of protein-protein interactions. Protein Sci 22:153-167.