Recombinant Escherichia coli Inner membrane protein ybjM (ybjM)

What is YejM and what is its basic structure?

YejM is an essential inner membrane protein found in Gram-negative bacteria such as E. coli and S. typhimurium. Structurally, it consists of five predicted helical transmembrane domains (5TM), followed by a positively charged arginine-rich (RR) linker region, and a C-terminal periplasmic domain (PD). The periplasmic domain has an α/β hydrolase fold architecture, consisting of alternating α-helices and β-sheets forming three layers. This domain shares structural similarities with members of the hydrolase superfamily including sulfatases, phospho-, mono-, and diesterases, and metalloenzymes .

What functional domains have been identified in YejM?

The YejM protein contains two distinct functional domains: the five transmembrane domain (5TM) anchored in the inner membrane, and the periplasmic domain (YejMPD) that extends into the periplasmic space. Research has shown that mutants lacking the periplasmic domain remain viable, whereas deletion of the 5TM domain is lethal for Gram-negative bacteria. Additionally, YejM contains a C-terminal (CT) domain of unknown function that shows structural similarity to proteins such as colicin and kinases like PLK1. This CT domain may serve as a site for oligomerization in some instances .

Why is YejM considered essential for bacterial survival?

YejM plays a critical role in maintaining bacterial membrane integrity and virulence. Strains lacking the YejMPD show increased sensitivity to temperature and antibiotics such as vancomycin. The protein is involved in several membrane remodeling pathways essential for cell survival, including:

• Regulation of LPS biosynthesis by preventing excessive degradation of LpxC

• Maintenance of phospholipid/LPS ratio in the outer membrane

• Promotion of cyclopropane ring formation of phospholipids during stress

• Contribution to membrane homeostasis pathways critical for bacterial survival

What enzymatic activity has been identified for YejM?

YejM functions as a metalloenzyme with phosphatase activity. Crystal structures of YejMPD have revealed an intact active site with bound metal ions (identified as Mg²⁺), located at the base of layers II and III of the hydrolase domain. Enzymatic assays confirm that YejM has magnesium-dependent phosphatase activity. The integrity of both the active site and the 5TM domain including the RR linker region are essential for this enzymatic function .

How does the active site of YejM compare to other metalloenzymes?

The active site coordination of YejM contains the conserved threonine 302 (Thr302) located at the base of layers II and III of the protein structure. Residues Asp268, Asn403, Arg451, and His468 are involved in metal, water, and substrate coordination. This active site architecture shares significant similarities with other metalloenzymes:

While there are differences between these active sites, the catalytically crucial nucleophilic threonine and surrounding residues are highly conserved across these metalloenzymes .

What is YejM's role in outer membrane remodeling?

YejM contributes to outer membrane remodeling through multiple mechanisms. It regulates LPS biosynthesis by preventing excessive degradation of LpxC, a key enzyme in the lipid A biosynthetic pathway. LpxC is a zinc-dependent metallo-amidase that performs the first committed step in LPS biosynthesis and is constantly degraded by the FtsH protease. YejM interferes with this degradation by interacting with YciM (also known as LapB), which normally presents LpxC to the FtsH protease. Through this mechanism, YejM plays a crucial role in maintaining proper LpxC levels and thus the appropriate phospholipid/LPS ratio in the outer membrane that is vital for cell survival .

What crystallization methods have been successful for YejM structural analysis?

The periplasmic domain of YejM from S. typhimurium has been successfully crystallized and its structure determined. Two crystal forms have been reported:

• Wild-type YejMPD: 2.35 Å resolution, space group P 32 2 1, cell dimensions a=b=113.80 Å, c=299.78 Å, α=β=90.00°, γ=120.00°

• YejMPD-F349A mutant: 2.05 Å resolution, space group P 21 21 21, cell dimensions a=121.45 Å, b=125.10 Å, c=182.75 Å, α=β=γ=90.00°

Data collection was performed at ALS BEAMLINE 4.2.2 with a wavelength of 1.00003 Å. The structures were refined to R-work/R-free values of 0.1963/0.2489 for wild-type and 0.1847/0.2270 for the F349A mutant .

How can the phosphatase activity of YejM be measured experimentally?

Phosphatase activity of YejM can be determined using enzymatic assays that measure magnesium-dependent phosphate release. The assay should be designed to:

• Test both intact YejM (with transmembrane domain) and the isolated periplasmic domain

• Include controls with and without magnesium to confirm metal dependence

• Assess the effects of mutations in key active site residues (particularly Thr302)

• Compare activity across different potential substrates relevant to membrane lipid metabolism

It's important to note that the integrity of both the active site and the 5TM domain, including the RR linker region, are essential for YejM enzymatic activity .

What genetic approaches can effectively study YejM function in vivo?

Several genetic approaches have proven effective for studying YejM function:

• Conditional knockouts: Since YejM is essential, conditional expression systems allow for controlled depletion of the protein to study effects on cell viability and membrane composition.

• Domain deletion analysis: Studies have shown that mutants lacking the periplasmic domain (YejMPD) remain viable but show increased sensitivity to temperature and antibiotics. This approach helps distinguish the functions of different protein domains.

• Suppressor mutation analysis: Strains lacking YejM have been rescued by overexpression of inactive or active forms of phosphopantetheinyl transferase (AcpT), suggesting genetic interactions that can be exploited to understand functional pathways.

• Synthetic lethal screening: Similar to studies with YciB, synthetic lethal partner identification can reveal genetic interactions that provide insight into YejM function .

How does YejM integrate with LPS biosynthesis regulation networks?

YejM functions as a key regulator in LPS biosynthesis by preventing excessive degradation of LpxC. It does this by interfering with the interaction between YciM (LapB) and the FtsH protease that would otherwise degrade LpxC. This regulation is critical for maintaining the appropriate phospholipid/LPS ratio in the outer membrane.

Future research should investigate:

• The molecular details of the YejM-YciM interaction

• How YejM's phosphatase activity relates to its role in LpxC regulation

• The signals that regulate YejM activity in response to changes in membrane composition

• Potential feedback mechanisms that link YejM function to LPS production rates

What is the relationship between YejM and other membrane homeostasis proteins?

YejM appears to function within a network of proteins involved in membrane homeostasis. Research has shown connections to:

• YciM (LapB): YejM interferes with YciM's ability to present LpxC for degradation

• FtsH protease: Indirectly regulated by YejM through its interaction with YciM

• AcpT (phosphopantetheinyl transferase): Overexpression can rescue YejM deficiency

• PhoPQ two-component system: YejM (also called PbgA in S. typhimurium) was discovered to play a role in PhoPQ-dependent changes to the outer membrane

Understanding the complete interaction network and signaling pathways connecting these proteins remains an important research direction. Techniques such as bacterial two-hybrid assays, co-immunoprecipitation, and cross-linking studies would be valuable for mapping these protein-protein interactions .

How might YejM function as a target for novel antimicrobial development?

YejM represents a potential target for new antimicrobial development for several reasons:

• It is essential for Gram-negative bacterial survival

• It has a well-defined enzymatic activity (phosphatase) that could be inhibited

• It plays a critical role in outer membrane integrity and antibiotic resistance

• Its structure has now been determined at high resolution

A structure-based drug design approach targeting the active site of YejM's periplasmic domain could yield inhibitors that disrupt outer membrane integrity, potentially increasing bacterial susceptibility to existing antibiotics or directly causing bacterial death. Combination approaches targeting both YejM and its interacting partners might be particularly effective at disrupting membrane homeostasis .

How does YejM relate phylogenetically to other bacterial membrane proteins?

Phylogenetic analysis of YejM's periplasmic domain reveals that it belongs to the larger phosphatase superfamily. Within this superfamily, YejM is more closely related to the LtaS protein found in Gram-positive bacteria and more distantly related to phosphoethanolamine (PEA) transferases such as EptA and MCR-1 in Gram-negative bacteria. This evolutionary relationship suggests that YejM may have diverged from a common ancestor shared with these other enzymes, adapting to specific functions in Gram-negative bacterial membrane homeostasis .

What structural features distinguish YejM from other similar metalloenzymes?

YejM shares the α/β hydrolase fold with other metalloenzymes but has several distinguishing features:

These distinctive features of YejM likely reflect its specialized role in Gram-negative bacterial membrane homeostasis and present opportunities for selective targeting in antimicrobial development.