The non-swapped monomeric structure of the arginine-binding protein from Thermotoga maritima

Abstract

Domain swapping is a widespread oligomerization process that is observed in a large variety of protein families. In the large superfamily of substrate-binding proteins, non-monomeric members have rarely been reported. The arginine-binding protein from Thermotoga maritima (TmArgBP), a protein endowed with a number of unusual properties, presents a domain-swapped structure in its dimeric native state in which the two polypeptide chains mutually exchange their C-terminal helices. It has previously been shown that mutations in the region connecting the last two helices of the TmArgBP structure lead to the formation of a variety of oligomeric states (monomers, dimers, trimers and larger aggregates). With the aim of defining the structural determinants of domain swapping in TmArgBP, the monomeric form of the P235GK mutant has been structurally characterized. Analysis of this arginine-bound structure indicates that it consists of a closed monomer with its C-terminal helix folded against the rest of the protein, as typically observed for substrate-binding proteins. Notably, the two terminal helices are joined by a single nonhelical residue (Gly235). Collectively, the present findings indicate that extending the hinge region and conferring it with more conformational freedom makes the formation of a closed TmArgBP monomer possible. On the other hand, the short connection between the helices may explain the tendency of the protein to also adopt alternative oligomeric states (dimers, trimers and larger aggregates). The data reported here highlight the importance of evolutionary control to avoid the uncontrolled formation of heterogeneous and potentially harmful oligomeric species through domain swapping.

The role of C-terminal helix in the conformational transition of an arginine binding protein

“…The structure of cs-TmArgBP ΔCTH refers to the structure of closed-state conformation of TmArgBP without the CTH (PDB: 6GGV). Recently, the crystal structure of a monomeric version of the protein in the closed-state conformation complexed with arginine has also been solved (PDB: 6SVF, which is referred as cs-TmArgBP CTH ) ( Smaldone et al, 2019 ). In this structure a proline residue occurring prior to CTH is mutated (P215GK).…”

“…Recently, a monomeric version of the arginine-bound closed-state conformation of TmArgBP has been solved. In this structure more flexibility is introduced in the loop prior to CTH by mutating PRO-215 with GLY(2 1 5)-LYS(2 1 6) enabling the CTH to fold against the same chain ( Smaldone et al, 2019 ).

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“…Biophysical investigations suggested this truncated version to be a better scaffold for arginine sensing while imposing a destabilization effect on the ligand-bound closed-state. In another attempt to generate the monomer a single proline residue prior to CTH was mutated and the crystal structure of this mutant was solved in a closed-state conformation bound to arginine ( Smaldone et al, 2019 ).…”

Development of a Protein Scaffold for Arginine Sensing Generated through the Dissection of the Arginine-Binding Protein from Thermotoga maritima

“…This protein presents a number of distinctive/unique features in the large class of SBP. Although substrate-binding proteins are generally monomeric, TmArgBP is essentially dimeric, with the concomitant presence, as minor components, of higher oligomerization states [ 14 , 15 , 16 ]. The crystallographic structure of the protein has highlighted that its dimer is stabilized by swapping of the terminal C -terminal helix [ 9 ] ( Figure 1 ).…”

“…We have exploited the extraordinary stability of TmArgBP to effectively manipulate the protein through mutation, truncation and dissection of its native structure [ 10 , 13 , 14 ]. In particular, we have shown that a stable monomeric form may be obtained either though site-directed mutagenesis [ 15 ] or through the deletion of the C -terminal end, the swapping element in the protein binding [ 10 ]. TmArgBP can be further manipulated by dissecting it into its constitutive D1 and D2 domains ( Figure 1 ).…”