Linkers or spacers are short amino acid sequences created in nature to separate multiple domains in a single protein. Most of them are rigid and function to prohibit unwanted interactions between the discrete domains. However, Gly-rich linkers are flexible, connecting various domains in a single protein without interfering with the function of each domain. The advent of recombinant DNA technology made it possible to fuse two interacting partners with the introduction of artificial linkers. Often, independent proteins may not exist as stable or structured proteins until they interact with their binding partner, following which they gain stability and the essential structural elements. Gly-rich linkers have been proven useful for these types of unstable interactions, particularly where the interaction is weak and transient, by creating a covalent link between the proteins to form a stable protein-protein complex. Gly-rich linkers are also employed to form stable covalently linked dimers, and to connect two independent domains that create a ligand-binding site or recognition sequence. The lengths of linkers vary from 2 to 31 amino acids, optimized for each condition so that the linker does not impose any constraints on the conformation or interactions of the linked partners. Various structures of covalently linked protein complexes have been described using X-ray crystallography, nuclear magnetic resonance and cryo-electron microscopy techniques. In this review, we evaluate several structural studies where linkers have been used to improve protein quality, to produce stable protein-protein complexes, and to obtain protein dimers.

SignificanceARE ABC-F genes have been found in numerous pathogen genomes and multi-drug resistance conferring plasmids. Further transmission will challenge the clinical use of many antibiotics. The development of improved ribosome-targeting therapeutics relies on the elucidation of the resistance mechanisms. Characterization of MsrE protein bound to the bacterial ribosome is first of its kind for ARE ABC-F members. Together with biochemical data, it sheds light on the ribosome protection mechanism by domain linker-mediated conformational change and displacement leading to drug release, suggesting a mechanism shared by other ARE ABC-F proteins. These proteins present an intriguing example of structure-function relationship and a medically relevant target of study as they collectively mediate resistance to the majority of antibiotic classes targeting the peptidyl-transferase center region.

In protein synthesis, elongation factor G (EF-G) facilitates movement of tRNA-mRNA by one codon, which is coupled to the ratchet-like rotation of the ribosome complex and is triggered by EF-G-mediated GTP hydrolysis. Here we report the structure of a pretranslocational ribosome bound to Thermus thermophilus EF-G trapped with a GTP analog. The positioning of the catalytic His87 into the active site coupled to hydrophobic-gate opening involves the 23S rRNA sarcin-ricin loop and domain III of EF-G and provides a structural basis for the GTPase activation of EF-G. Interactions of the hybrid peptidyl-site-exit-site tRNA with ribosomal elements, including the entire L1 stalk and proteins S13 and S19, shed light on how formation and stabilization of the hybrid tRNA is coupled to head swiveling and body rotation of the 30S as well as to closure of the L1 stalk.

Neuromodulin (Nm) and neurogranin (Ng) are neuron-specific substrates of protein kinase C (PKC). Their interactions with Calmodulin (CaM) are crucial for learning and memory formation in neurons. Here, we report the structure of IQ peptides (24aa) of Nm/Ng complexed with CaM and their functional studies with full-length proteins. Nm/Ng and their respective IQ peptides are intrinsically unstructured; however, upon binding with CaM, IQ motifs adopt a helical conformation. Ser41 (Ser36) of Nm (Ng) is located in a negatively charged pocket in the apo CaM and, when phosphorylated, it will repel Nm/Ng from CaM. These observations explain the mechanism by which PKC-induced Ser phosphorylation blocks the association of Nm/Ng with CaM and interrupts several learning- and memory-associated functions. Moreover, the present study identified Arg as a key CaM interacting residue from Nm/Ng. This residue is crucial for CaM-mediated function, as evidenced by the inability of the Ng mutant (Arg-to-Ala) to potentiate synaptic transmission in CA1 hippocampal neurons.

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BPI-inducible protein A (BipA) is a member of the family of ribosome-dependent translational GTPase (trGTPase) factors along with elongation factors G and 4 (EF-G and EF4). Despite being highly conserved in bacteria and playing a critical role in coordinating cellular responses to environmental changes, its structures (isolated and ribosome bound) remain elusive. Here, we present the crystal structures of apo form and GTP analog, GDP, and guanosine-3′,5′-bisdiphosphate (ppGpp)-bound BipA. In addition to having a distinctive domain arrangement, the C-terminal domain of BipA has a unique fold. Furthermore, we report the cryo-electron microscopy structure of BipA bound to the ribosome in its active GTP form and elucidate the unique structural attributes of BipA interactions with the ribosome and A-site tRNA in the light of its possible function in regulating translation.

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