Positive Selection Dictates the Choice between Kinetic and Thermodynamic Protein Folding and Stability in Subtilases

Subbian, Ezhilkani; Yabuta, Yukihiro; Shinde, Ujwal

doi:10.1021/bi048397x

Cited by 30 publications

(50 citation statements)

References 77 publications

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“…Furthermore, a region of the S. thermophilus prtS propeptide domain is duplicated. This domain functions as an intramolecular chaperone in the subtilisin protease family (39), suggesting that this change is required to direct correct folding of PrtS in its new host. These modifications indicate that prtS could evolves rapidly in S. thermophilus, possibly under positive selection, contrary to the surrounding region that contains duplicates of genes that already exist in S. thermophilus.…”

Section: Discussionmentioning

confidence: 99%

Emergence of a Cell Wall Protease in the Streptococcus thermophilus Population

Delorme

Bartholini

Bolotine

et al. 2010

Appl Environ Microbiol

106

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Streptococcus thermophilus is perceived as a recently emerged food bacterium that evolved from a commensal ancestor by loss and gain of functions. Here, we provide data allowing a better understanding of this evolutionary scheme. A multilocus sequence typing approach that we developed showed that S. thermophilus diverges significantly from its potential ancestors of the salivarius group and displays a low level of allelic variability, confirming its likely recent emergence. An analysis of the origin and dissemination of the prtS gene was carried out within this evolutionary scheme. This gene encodes a protease that allows better growth in milk by facilitating casein breakdown to supply amino acids. The S. thermophilus protease exhibits 95% identity to the animal Streptococcus suis protein PrtS. Genomic analysis showed that prtS is part of an island flanked by two tandem insertion sequence elements and containing three other genes which present the best identities and synteny with the S. suis genome. These data indicate a potential origin for this "ecological" island in a species closely related to S. suis. The analysis of the distribution of the prtS gene in S. thermophilus showed that the gene is infrequent in historical collections but frequent in recent industrial ones. Moreover, this "ecological" island conferring an important metabolic trait for milk adaptation appears to have disseminated by lateral transfer in the S. thermophilus population. Taken together, these data support an evolutionary scheme of S. thermophilus where gene acquisition and selection by food producers are determining factors. The source and impact of genes acquired by horizontal gene transfer on the physiology and safety of strains should be addressed.

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Section: Discussionmentioning

confidence: 99%

Emergence of a Cell Wall Protease in the Streptococcus thermophilus Population

Delorme

Bartholini

Bolotine

et al. 2010

Appl Environ Microbiol

106

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“…The circular dichroism (CD) spectra of proISP S250A and NΔ 18 -ISP were almost identical ( Fig. S2A) and characteristic of proteins with a subtilisin-like fold (20,21,24). This indicates that there is no major change in the overall structure of ISP on processing.…”

Section: Proteolytic Processing Of Isp Has Little Effect On Overall Tmentioning

confidence: 90%

“…However, little is known regarding the crucial feature of how their activity is regulated posttranslationally within the cell, where control of protease activity is vital to prevent the untimely breakdown of crucial cellular protein components. This is exemplified by the harmful effects of intracellular expression of bacilli ESPs to the host cell (20).…”

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confidence: 99%

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Regulation of an intracellular subtilisin protease activity by a short propeptide sequence through an original combined dual mechanism

Gamble

Künze

Dodson

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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A distinct class of the biologically important subtilisin family of serine proteases functions exclusively within the cell and forms a major component of the bacilli degradome. However, the mode and mechanism of posttranslational regulation of intracellular protease activity are unknown. Here we describe the role played by a short N-terminal extension prosequence novel amongst the subtilisins that regulates intracellular subtilisin protease (ISP) activity through two distinct modes: active site blocking and catalytic triad rearrangement. The full-length proenzyme (proISP) is inactive until specific proteolytic processing removes the first 18 amino acids that comprise the N-terminal extension, with processing appearing to be performed by ISP itself. A synthetic peptide corresponding to the N-terminal extension behaves as a mixed noncompetitive inhibitor of active ISP with a K i of 1 μM. The structure of the processed form has been determined at 2.6 Å resolution and compared with that of the full-length protein, in which the N-terminal extension binds back over the active site. Unique to ISP, a conserved proline introduces a backbone kink that shifts the scissile bond beyond reach of the catalytic serine and in addition the catalytic triad is disrupted. In the processed form, access to the active site is unblocked by removal of the N-terminal extension and the catalytic triad rearranges to a functional conformation. These studies provide a new molecular insight concerning the mechanisms by which subtilisins and protease activity as a whole, especially within the confines of a cell, can be regulated.posttranslational modification | protease regulation | proprotein activation | protease structure S ubtilisins are serine endopeptidases ubiquitous in nature, spread across the eubacteria, archaebacteria, eukaryotes, and viruses (1). They play a variety of important biological roles that include specific posttranslational processing of hormones (2, 3), virulence and infection factors in various pathogens (4, 5), and as nonspecific digestive proteases (1). The distinctive primary structure features common to the majority of subtilisins, and typified by the bacilli extracellular subtilisins (ESP), comprise a N-terminal signal sequence with an adjacent prodomain required for correct folding of the mature, catalytic domain (6-8) (Fig. 1A). Both the signal sequence and the prodomain are posttranslationally removed, the latter autocatalytically. The bacilli ESPs have a broad substrate specificity that suites their role as scavenging proteases, and their robustness to harsh environments coupled with extensive protein engineering has resulted in their exploitation by industry for a variety of applications, in particular as an active ingredient in laundry detergents (6, 7, 9, 10). The ESPs have proved excellent paradigms for understanding key molecular features of proteins, including enzyme catalysis (11) and protein folding (6-8).The intracellular subtilisins (ISPs) are a distinctive class found in many different bacilli and re...

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“…Side chains were modeled using SCWRL3.0, a program for predicting protein side-chain conformations for a given backbone. The best models were minimized using CharmM and then validated as previously described (Subbian et al, 2004). The structures were visualized using PYMOL version 0.99.…”

Section: Molecular Modeling Using Rosetta Softwarementioning

confidence: 99%

Both sequence and context are important for flagellar targeting of a glucose transporter

Tran¹,

Rodriguez‐Contreras²,

Shinde³

et al. 2012

Journal of Cell Science

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SummaryMany of the cilia-and flagella-specific integral membrane proteins identified to date function to sense the extracellular milieu, and there is considerable interest in defining pathways for targeting such proteins to these sensory organelles. The flagellar glucose transporter of Leishmania mexicana, LmxGT1, is targeted selectively to the flagellar membrane, whereas two other isoforms, LmxGT2 and LmxGT3, are targeted to the pellicular plasma membrane of the cell body. To define the flagellar targeting signal, deletions and point mutations were generated in the N-terminal hydrophilic domain of LmxGT1, which mediates flagellar localization. Three amino acids, N95-P96-M97, serve critical roles in flagellar targeting, resulting in strong mistargeting phenotypes when mutagenized. However, to facilitate flagellar targeting of other non-flagellar membrane proteins, it was necessary to attach a larger region surrounding the NPM motif containing amino acids 81-113. Molecular modeling suggests that this region might present the critical NPM residues at the surface of the N-terminal domain. It is likely that the NPM motif is recognized by currently unknown protein-binding partners that mediate flagellar targeting of membrane-associated proteins.

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Positive Selection Dictates the Choice between Kinetic and Thermodynamic Protein Folding and Stability in Subtilases

Cited by 30 publications

References 77 publications

Emergence of a Cell Wall Protease in the Streptococcus thermophilus Population

Emergence of a Cell Wall Protease in the Streptococcus thermophilus Population

Regulation of an intracellular subtilisin protease activity by a short propeptide sequence through an original combined dual mechanism

Both sequence and context are important for flagellar targeting of a glucose transporter

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