Small-angle neutron scattering studies suggest the mechanism of BinAB protein internalization

This study reports a solution structural investigation of the Cqm1–BinB protein complex using ‘contrast-matched’ small-angle neutron scattering (SANS). The SANS data clearly reveal that BinB binds to the receptor Cqm1 and alters its oligomeric status from a dimer to a monomer.


S1.1. Introduction of point mutations in the pET28a(+)-binB construct using overlap-extension PCR
The recombinant pET28a(+)-binB construct encoding full length BinB protein (ISPC-8) was used as a construct to introduce two point mutations in the binB gene by overlap extension PCR (OE-PCR) translating to two substitutions in BinB protein-His109Pro and Pro274Ser.
OE-PCR based mutagenesis was performed in a two-step PCR procedure. In the first step, two double stranded DNA fragments with overlapping ends carrying the point mutation for His109Pro substitution were generated through PCR using primers 5'-CCGGCGTAGAGGATCGAGAT-3' and 5'-GTTATTATCCAAATAAGTAGGTGCTTCATCAAAAAC-3' (His109Pro product 1) and primers, 5'-GTTTTTGATGAAGCACCTACTTATTTGGATAATAAC-3' and 5'-TAGTTATTGCTCAGCGGTGG-3' (His109Pro product 2). The expected fragments as observed from gel were excised and purified using gel purification kit (Qiagen). In the second step, His109Pro products 1 and 2 were mixed in a 1: 1 ratio and amplified in a modified PCR reaction consisting of 10 cycles in the absence of any primers to produce full length double stranded DNA. Following 10 cycles, extreme end primers, 5'-CCGGCGTAGAGGATCGAGAT-3' and 5'-TAGTTATTGCTCAGCGGTGG-3' were added and the PCR reaction was continued for 25 more cycles to amplify the full length binB gene with point mutation for His109Pro substitution. The binB gene after this stage was used as template for introducing second point mutation for P274S substitution. Primers sets, 5'-ATATACATATGTGCGATTCAAAAGACAATTCTGG-3' and 5'-GCAGGTATAATTTGTGACCATAATTGATGCCAG-3', and 5'-CTGGCATCAATTATGGTCACAAATTATACCTGC and 5'-TATAGGATCCTCATTACTGGTTAATTTTAGG-3' were used to amplify the two PCR fragments with overlapping ends carrying the point mutation for Pro274Ser substitution. In the second step, the PCR products were mixed in a 1: 1 ratio and amplified in a modified PCR reaction consisting of 10 cycles in the absence of any primers to produce full length double stranded mutagenic DNA. Following 10 cycles, binB gene specific primers with NdeI and BamHI restriction sites, 5'-ATATACATATGTGCGATTCAAAAGACAATTCTGG -3' and 5'-TATAGGATCCTCATTACTGGTTAATTTTAGG -3' were added and the PCR reaction was continued for 25 more cycles to amplify the complete gene. The modified binB gene with two point mutations for two substitutions (His109Pro and Pro274Ser) was isolated and purified using gel purification kit (Qiagen). The point mutations were confirmed by nucleotide sequencing. The modified binB gene was then cloned into pET28a(+) expression vector using NdeI (NEB) and BamHI (NEB) restriction sites and T4 DNA ligase (NEB) and the construct was transformed into E. coli XL-10 gold competent cells under kanamycin (50 μg/ml) selection. Positively transformed colonies were selected through colony PCR using Taq DNA polymerase (Invitrogen). The modified pET28a(+)-binB construct was isolated IUCrJ (2020). 7, doi:10.1107/S2052252519017159 Supporting information, sup-2 using plasmid purification kit (Qiagen) and transformed into BL21star(DE3) competent cells for protein expression.

S1.2. Expression and purification of rationally engineered BinB protein
For protein expression, overnight grown culture of E. coli harbouring pET28a(+)-binB construct was used to inoculate 1L LB medium supplemented with kanamycin (50 µg/ml) and grown further at 37 o C at 150 rpm. At cell density of ~0.7 (OD600), the temperature was lowered down to 18 o C and protein expression was induced with 0.5 mM IPTG. Cells were grown further overnight at 18 o C at 150 rpm.
Later, cells were harvested at ~6000 rpm for 5 min and resuspended in lysis buffer (

S2. Expression and purification of deuterated BinB protein
To facilitate E. coli cell growth and dBinB protein expression in D2O, a three-step adaptive approach was followed to adapt E.coli cells to D2O based growth medium: LB in H2O to LB in D2O to M9+ in D2O. A 5 ml LB/H2O culture (contained in a 100 ml flask) was started from a fresh agar plate of E. coli BL21star (DE3) harbouring pET28a(+)-binB construct. Cells were grown for 3 h at 37 °C at 150 rpm following which 500 μl from LB/H2O culture were inoculated to 5 ml of fresh LB/D2O medium (contained in a 100 ml flask). The cells were grown further at 37 °C for about 5 hr at 150 rpm till the cell density (OD600) reached ~ 0.5 to 1. The overnight pre-culture was started by transferring the 5 ml of LB/D2O culture into a 25 ml M9+/ D2O medium (contained in a 250 ml flask). Cells were grown overnight at 37 °C at 150 rpm. The 25 ml pre-culture was then inoculated into 250 ml of M9+/ D2O medium (contained in a 1L flask) and allowed to grow at 37 °C at 150 rpm till OD600 reached ~0.7. The shaker temperature was then reduced to 20 °C and protein expression was induced by adding 1mM IPTG. Cells were grown further for an extended period of 48 h at 20 °C before harvesting. Cells were harvested and lysed following the protocol similar for hydrogenous BinB protein (details in Supplementary Method S1.2) except that all the buffers used for deuterated BinB protein were made in 100% D2O. The deuterated protein was purified to homogeneity using immobilized metal ion affinity chromatography.