Search

The maltose-binding protein (MBP) is one of the most frequently used protein tags due to its capacity to stabilize, solubilize and even crystallize recombinant proteins that are fused to it. Given that MBP is thought to be a highly stable monomeric protein with known characteristics, fused passenger proteins are often studied without being cleaved from MBP. Here we report that a commonly used engineered MBP version (mutated to lower its surface entropy) can form interlaced dimers when fused to short protein sequences derived from the focal adhesion kinase (FAK) or the homologous protein tyrosine kinase 2 (PYK2). These MBP dimers still bind maltose and can interconvert with monomeric forms in vitro under standard conditions despite a contact surface of more than 11,000 Å2. We demonstrate that both the mutations in MBP and the fused protein sequences were required for dimer formation. The FAK and PYK2 sequences are less than 40% identical, monomeric, and did not show specific interactions with MBP, suggesting that a variety of sequences can promote this MBP dimerization. MBP dimerization was abrogated by reverting two of the eight mutations introduced in the engineered MBP. Our results provide an extreme example for induced reversible domain-swapping, with implications for protein folding dynamics. Our observations caution that passenger-promoted MBP dimerization might mislead experimental characterization of the fused protein sequences, but also suggest a simple mutation to stop this phenomenon. [ABSTRACT FROM AUTHOR]

As opposed to typical bacteria exhibiting chemotaxis towards low-molecular-weight substances, such as amino acids and mono/oligosaccharides, gram-negative Sphingomonas sp. strain A1 shows chemotaxis towards alginate and pectin polysaccharides. To identify the mechanism of chemotaxis towards macromolecules, a genomic fragment was isolated from the wild-type strain A1 through complementation with the mutant strain A1-M5 lacking chemotaxis towards pectin. This fragment contained several genes including sph1118. Through whole-genome sequencing of strain A1-M5, sph1118 was found to harbour a mutation. In fact, sph1118 disruptant lost chemotaxis towards pectin, and this deficiency was recovered by complementation with wild-type sph1118. Interestingly, the gene disruptant also exhibited decreased pectin assimilation. Furthermore, the gene product SPH1118 was expressed in recombinant E. coli cells, purified and characterised. Differential scanning fluorimetry and UV absorption spectroscopy revealed that SPH1118 specifically binds to pectin with a dissociation constant of 8.5 μM. Using binding assay and primary structure analysis, SPH1118 was predicted to be a periplasmic pectin-binding protein associated with an ATP-binding cassette transporter. This is the first report on the identification and characterisation of a protein triggering chemotaxis towards the macromolecule pectin as well as its assimilation.

BtuCD-F is an ABC transporter that mediates cobalamin uptake into Escherichia coli. Early in vivo data suggested that BtuCD-F might also be involved in the uptake of cobinamide, a cobalamin precursor. However, neither was it demonstrated that BtuCD-F indeed transports cobinamide, nor was the structural basis of its recognition known. We synthesized radiolabeled cyano-cobinamide and demonstrated BtuCD-catalyzed in vitro transport, which was ATP- and BtuF-dependent. The crystal structure of cobinamide-bound BtuF revealed a conformational change of a tryptophan residue (W66) in the substrate binding cleft compared to the structure of cobalamin-bound BtuF. High-affinity binding of cobinamide was dependent on W66, because mutation to most other amino acids substantially reduced binding. The structures of three BtuF W66 mutants revealed that tight packing against bound cobinamide was only provided by tryptophan and phenylalanine, in line with the observed binding affinities. In vitro transport rates of cobinamide and cobalamin were not influenced by the substitutions of BtuF W66 under the experimental conditions, indicating that W66 has no critical role in the transport reaction. Our data present the molecular basis of the cobinamide versus cobalamin specificity of BtuCD-F and provide tools for in vitro cobinamide transport and binding assays. [ABSTRACT FROM AUTHOR]