Crystal structure of basal pilin SpaE reveals the molecular basis of its incorporation in the lactobacillar SpaFED pilus

Crystal structure of the atypically adhesive SpaB basal pilus subunit: Mechanistic insights about its incorporation in lactobacillar SpaCBA pili

“…S1 , and Table S1 ). Consequently, there is a good possibility that the K–N isopeptide interaction forms more slowly in GG-SpaB FL much like what was observed previously in some other pilins ( Kang et al., 2014 ; Megta et al., 2019 ). Here, it is also worth mentioning that the key residues needed for forming internal and intermolecular isopeptide bonds are well conserved in both a sequence and structural alignment between GG-SpaB and the closely related backbone GG-SpaA pilin ( Fig.…”

“…For some time, we have delved into solving the crystal structures of the pilin ( Chaurasia et al., 2016 , 2018 ; Kant et al., 2016 , 2020 ; Kumar Megta et al., 2019 ; Megta et al., 2019 ; Mishra et al., 2017 ; Singh et al., 2013 ) and sortase ( Pratap et al., 2019 ) proteins from Lactobacillus rhamnosus GG, a strongly adapted gut-transient probiotic strain and one of just a few known commensal bacteria with sortase-dependent pili ( Kankainen et al., 2009 ; Kant et al., 2014 ; Lebeer et al., 2009 ; Turroni et al., 2013 ; Yu et al., 2015 ). L. rhamnosus GG contains the spaCBA operon ( spaC - spaB - spaA - srtC1 ) that produces the so-called SpaCBA pilus, which is comprised of the tip SpaC, basal SpaB, and backbone SpaA pilins ( Kankainen et al., 2009 ; Reunanen et al., 2012 ).…”

SpaB, an atypically adhesive basal pilin from the lactobacillar SpaCBA pilus: crystallization and X-ray diffraction analysis

“…As similarly pursued for many other sortase-dependent pilus types (Krishnan, 2015), and in order to better understand their molecular assembly and functional processes, we have initiated an X-ray crystallographic study of the various pilin subunits composing the L. rhamnosus GG SpaCBA and SpaFED pili. Thus far, we have reported the crystallization and preliminary X-ray diffraction analysis of the SpaA (Singh et al, 2013), SpaD (Chaurasia et al, 2015), SpaE (Mishra et al, 2017) and SpaC (Kant et al, 2016) pilins, along with the subsequent tertiary-structure determination of the former three proteins (Chaurasia et al, 2016(Chaurasia et al, , 2018Megta et al, 2019). Our structural findings revealed that SpaA, SpaD and SpaE resemble other Gram-positive pilins by having a modular domain arrangement that consists of the so-called CnaA and CnaB domains (Kang & Baker, 2012;Krishnan, 2015), both of which represent immunoglobulin (Ig)-like fold variants of the staphylococcal collagen adhesin (Cna) (Deivanayagam et al, 2000;Symersky et al, 1997).…”

“…As this latter conformational state had never been observed previously, we were also able to conceive and put forth the three-stage 'expose-ligate-seal' model (Chaurasia et al, 2018), which provides a mechanistic explanation of the docking and assembly of backbone pilins into a sortasedependent pilus. Our recent crystal structure solution of SpaE (Megta et al, 2019) shows that this basal pilin is a two-domain protein (CnaB-CnaB) with two K-N isopeptide bonds, in which the N-domain contains a slow-formed bond. Interestingly, we research communications also found that the expose-ligate-seal mechanism seems to aptly describe how SpaE might dock at the base of the SpaD backbone polymer during pilus assembly.…”

“…The focus of our present efforts is to obtain X-ray diffraction-quality crystals of SpaB for its subsequent structural determination. However, since compared with backbone pilins, for example, the ancillary basal subunits tend to be less stable proteins when in a recombinant form, their crystallization has been far more challenging and difficult to achieve, and thus is reflected by only a small number of solved basal pilin structures (Krishnan et al, 2007;Linke et al, 2010;Megta et al, 2019;Shaik et al, 2014). In this article, we report how we overcame this problem by implementing a crystallization strategy that combined the use of an SpaB protein construct with a truncated flexible C-terminal tail region along with the use of surface lysine methylation (SLM) and magnesium additives during crystal growth.…”