4.H.1 The Lysyl Phosphatidylglycerol Synthase/Flippase (MprF) Family

Phospholipids are synthesized at the inner leaflet of the bacterial cytoplasmic membrane but have to be translocated to the outer leaflet to maintain membrane lipid bilayer composition and structure. MprF is a large integral membrane protein found in several prokaryotic phyla, the C-terminus of which modifies phosphatidylglycerol (PG) with lysine or alanine to modulate the membrane surface charge and, as a consequence, confer resistance to cationic antimicrobial agents such as daptomycin (Ernst and Peschel 2011). In addition, MprF is a flippase for the resulting lipids, Lys-PG or Ala-PG. Ernst et al. 2015 demonstrated that the flippase activity resides in the N-terminal 6 to 8 transmembrane segments of the Staphylococcus aureus MprF and that several conserved, charged amino acids and a proline residue are crucial for flippase function. MprF protects S. aureus against the membrane-active antibiotic daptomycin only when both domains are present, but the two parts do not need to be covalently linked and can function in trans (Hebecker et al. 2015). The Lys-PG synthase and flippase domains were each found to homo-oligomerize and also to interact with each other, which illustrates how the two functional domains may act together. Moreover, full-length MprF proteins formed oligomers, indicating that MprF functions as a dimer or larger oligomer (Ernst et al. 2015). The data reveal how bacterial phospholipid flippases may function in the context of lipid biosynthetic processes. The coupling of lysyl transfer to lipid export is hypothetical.

Bacterial cytoplasmic membranes have to cope with membrane-damaging agents such as cationic antimicrobial peptides (CAMPs) produced, for example, by competing bacteria producing bacteriocins, secreted by eukaryotic host cells (defensins), or used as antimicrobial therapy (daptomycin). MprF proteins are found in many Gram-positive, Gram-negative, and archaeal commensals or pathogens and confers resistance to CAMPs by modifying anionic phospholipids with amino acids, thereby compromising the membrane interaction of CAMPs. Ernst et al. 2015 described how MprF does not only modify phospholipids but uses an additional, distinct domain for translocating the resulting lysinylated phospholipids to the outer leaflet of the membrane. They revealed critical details for the structure and function of MprF (Ernst et al. 2015). Moreover, gain-of-function mutations in MprF confer specific daptomycin resistance (Ernst et al. 2018).

Lysyl-tRNA synthetases are highly conserved enzymes that function in mRNA translation (Motzik et al. 2013). These synthetases have gained several functions in addition to protein synthesis, playing roles in HIV replication, cytokine-like signaling, and transport of proteins Motzik et al. 2013). Some of them are around 600 aas in length and possess a hydrophobic N-terminal domain (Pfam: PF16995) with 6-7 putative TMSs in addition to an uncharacterized hydrophilic domain at their C-termini (Pfam: PF09924). Some membeers of the family also have an MFS (TC# 2.A.1) domain.

The LysS family shows extensive sequence similarity to that of The Glycosyl Transferase 2 (GT2) Family (TC# 4.D.2) as well as an N-terminal duplicated region of the Mg²⁺ ATPases with TC #s 3.A.3.4.3 and 3.A.3.4.4. The GT2 (COG0392) family has been implicated in several distinct functions including drug resistance and various enzymatic activities. Most members have about 8 - 10 TMSs plus a ~340aa C-terminal hydrophilic domain that may have catalytic activity (see above). These proteins are implicated in glycosyl transfer with concomitant export across the membrane. They show very significant similarity to extra N-terminal domains in 3.A.3.4.3 and 4. These two putative Mg²⁺ P-type ATPases (but not others in this TC subfamily) have N-terminal duplications probably of 6 + 4 TMSs, or parts of them, and this duplicated region is shared by membres of the GT2 family as well as the MprF family (4.H.1). In all these families, this region may function to flip the substrate(s) from the inner surface of the membrane to the outer surface of it. The C-terminal domains in 2.A.1.3.43 (MFS) can be found in some of these proteins (unpublished results).

4.H.1 The Lysyl Phosphatidylglycerol Synthase/Flippase (MprF) Family Phospholipids are synthesized at the inner leaflet of the bacterial cytoplasmic membrane but have to be translocated to the outer leaflet to maintain membrane lipid bilayer composition and structure. MprF is a large integral membrane protein found in several prokaryotic phyla, the C-terminus of which modifies phosphatidylglycerol (PG) with lysine or alanine to modulate the membrane surface charge and, as a consequence, confer resistance to cationic antimicrobial agents such as daptomycin (Ernst and Peschel 2011). In addition, MprF is a flippase for the resulting lipids, Lys-PG or Ala-PG. Ernst et al. 2015 demonstrated that the flippase activity resides in the N-terminal 6 to 8 transmembrane segments of the Staphylococcus aureus MprF and that several conserved, charged amino acids and a proline residue are crucial for flippase function. MprF protects S. aureus against the membrane-active antibiotic daptomycin only when both domains are present, but the two parts do not need to be covalently linked and can function in trans (Hebecker et al. 2015). The Lys-PG synthase and flippase domains were each found to homo-oligomerize and also to interact with each other, which illustrates how the two functional domains may act together. Moreover, full-length MprF proteins formed oligomers, indicating that MprF functions as a dimer or larger oligomer (Ernst et al. 2015). The data reveal how bacterial phospholipid flippases may function in the context of lipid biosynthetic processes. The coupling of lysyl transfer to lipid export is hypothetical. Bacterial cytoplasmic membranes have to cope with membrane-damaging agents such as cationic antimicrobial peptides (CAMPs) produced, for example, by competing bacteria producing bacteriocins, secreted by eukaryotic host cells (defensins), or used as antimicrobial therapy (daptomycin). MprF proteins are found in many Gram-positive, Gram-negative, and archaeal commensals or pathogens and confers resistance to CAMPs by modifying anionic phospholipids with amino acids, thereby compromising the membrane interaction of CAMPs. Ernst et al. 2015 described how MprF does not only modify phospholipids but uses an additional, distinct domain for translocating the resulting lysinylated phospholipids to the outer leaflet of the membrane. They revealed critical details for the structure and function of MprF (Ernst et al. 2015). Moreover, gain-of-function mutations in MprF confer specific daptomycin resistance (Ernst et al. 2018). Lysyl-tRNA synthetases are highly conserved enzymes that function in mRNA translation (Motzik et al. 2013). These synthetases have gained several functions in addition to protein synthesis, playing roles in HIV replication, cytokine-like signaling, and transport of proteins Motzik et al. 2013). Some of them are around 600 aas in length and possess a hydrophobic N-terminal domain (Pfam: PF16995) with 6-7 putative TMSs in addition to an uncharacterized hydrophilic domain at their C-termini (Pfam: PF09924). Some membeers of the family also have an MFS (TC# 2.A.1) domain. The LysS family shows extensive sequence similarity to that of The Glycosyl Transferase 2 (GT2) Family (TC# 4.D.2) as well as an N-terminal duplicated region of the Mg²⁺ ATPases with TC #s 3.A.3.4.3 and 3.A.3.4.4. The GT2 (COG0392) family has been implicated in several distinct functions including drug resistance and various enzymatic activities. Most members have about 8 - 10 TMSs plus a ~340aa C-terminal hydrophilic domain that may have catalytic activity (see above). These proteins are implicated in glycosyl transfer with concomitant export across the membrane. They show very significant similarity to extra N-terminal domains in 3.A.3.4.3 and 4. These two putative Mg²⁺ P-type ATPases (but not others in this TC subfamily) have N-terminal duplications probably of 6 + 4 TMSs, or parts of them, and this duplicated region is shared by membres of the GT2 family as well as the MprF family (4.H.1). In all these families, this region may function to flip the substrate(s) from the inner surface of the membrane to the outer surface of it. The C-terminal domains in 2.A.1.3.43 (MFS) can be found in some of these proteins (unpublished results).
This family belongs to the Glycosyl Transferase/Transporter (GTT) Superfamily.
References:
Ernst, C.M. and A. Peschel. (2011). Broad-spectrum antimicrobial peptide resistance by MprF-mediated aminoacylation and flipping of phospholipids. Mol. Microbiol. 80: 290-299.
Ernst, C.M., C.J. Slavetinsky, S. Kuhn, J.N. Hauser, M. Nega, N.N. Mishra, C. Gekeler, A.S. Bayer, and A. Peschel. (2018). Gain-of-Function Mutations in the Phospholipid Flippase MprF Confer Specific Daptomycin Resistance. MBio 9:.
Ernst, C.M., S. Kuhn, C.J. Slavetinsky, B. Krismer, S. Heilbronner, C. Gekeler, D. Kraus, S. Wagner, and A. Peschel. (2015). The lipid-modifying multiple peptide resistance factor is an oligomer consisting of distinct interacting synthase and flippase subunits. MBio 6:.
Hebecker, S., J. Krausze, T. Hasenkampf, J. Schneider, M. Groenewold, J. Reichelt, D. Jahn, D.W. Heinz, and J. Moser. (2015). Structures of two bacterial resistance factors mediating tRNA-dependent aminoacylation of phosphatidylglycerol with lysine or alanine. Proc. Natl. Acad. Sci. USA 112: 10691-10696.
Helmstetter, F., P. Arnold, B. Höger, L.M. Petersen, and E. Beitz. (2019). Formate-nitrite transporters carrying nonprotonatable amide amino acids instead of a central histidine maintain pH-dependent transport. J. Biol. Chem. 294: 623-631.
Kristian, S.A., M. Dürr, J.A. Van Strijp, B. Neumeister, and A. Peschel. (2003). MprF-mediated lysinylation of phospholipids in Staphylococcus aureus leads to protection against oxygen-independent neutrophil killing. Infect. Immun. 71: 546-549.
Motzik, A., H. Nechushtan, S.Y. Foo, and E. Razin. (2013). Non-canonical roles of lysyl-tRNA synthetase in health and disease. Trends Mol Med 19: 726-731.
Peschel, A., R.W. Jack, M. Otto, L.V. Collins, P. Staubitz, G. Nicholson, H. Kalbacher, W.F. Nieuwenhuizen, G. Jung, A. Tarkowski, K.P. van Kessel, and J.A. van Strijp. (2001). Staphylococcus aureus resistance to human defensins and evasion of neutrophil killing via the novel virulence factor MprF is based on modification of membrane lipids with l-lysine. J Exp Med 193: 1067-1076.
Slavetinsky, C.J., A. Peschel, and C.M. Ernst. (2012). Alanyl-phosphatidylglycerol and lysyl-phosphatidylglycerol are translocated by the same MprF flippases and have similar capacities to protect against the antibiotic daptomycin in Staphylococcus aureus. Antimicrob. Agents Chemother. 56: 3492-3497.
Examples:
TC#	Name	Organismal Type	Example
4.H.1.1.1	The 14 TMS FmtC (MprF: Multiple peptide resistance factor) protein of 840 aas, involved in methicillin and daptomycin resistance. Residues 1-550 comprise a 14TMS MFS permease domain while residues 551-840 comprise a "phosphatidylglycerol lysyl transferase" (or synthetase) domain (DUF2156), also called "lysyl cardiolipid synthase" (Oku et al. 2004; Staubitz et al. 2004; Ernst et al. 2009). FmtC may be distantly related to lysyl-tRNA synthetases (TC# 9.B.111). Similar to 2.A.1.3.44 in all these respects. Ernst et al. 2015 and Slavetinsky et al. 2012 have reported that TMSs 1-6 of FmtC of S. aureus is a flippase for lysylinated phosphatidyl glycerol, and that the entire system is a dimer.	Bacteria	*FmtC of Staphylococcus aureus* (D1QCY9)**

4.H.1.1.10	Bifunctional lysylphosphatidylglycerol flippase/synthetase, MprF, of 850 aas and 15 or 16 putative TMSs in a 14 or 15 (N-terminal) +1 (C-terminal) TMS arrangement.		*MprF of Candidatus Brocadia* sp.**

4.H.1.1.11	Lysyl tRNA synthetase of 601 aas and 7 or 8 putative TMSs in an apparent 6 + 1 TMS arrangement, possibly with an additional C-terminal TMS, LysS (shows sequence similarity to MFS permease-like proteins with TC#s 4.H.1 and 2.A.1.3.9).	Bacteria	*LysSl of Streptomyces coelicolor* (Q9X8H7)**

4.H.1.1.12	Lysyl tRNA synthetase, LysS2 (shows extensive sequence similarity to MFS permease-like proteins with TC#s 4.H.1.1.	Bacteria	*LysS2 of Streptomyces coelicolor* (O69916)**

4.H.1.1.13	DUF2156 domain-containing protein of 608 aas and 6 putative N-terminal TMSs, possibly followed by more, including one at the C-terminus.		DUF2156 protein of Kitasatospora cheerisanensis

4.H.1.1.14	DUF2156 domain-containing protein of 584 aas and 7 N-terminal TMSs.		DUF2156 of Frankia coriariae

4.H.1.1.15	Lysine-tRNA ligase of 909 aas and 6 central putatve TMSs (residues 401 to 560) with a C-terminal ligase domain. The transmembrane domain of this protein shows poor sequence similarity with other members of this family and with MFS porters.		tRNA ligase of Solirubrobacter pauli

4.H.1.1.2	Putative oxacillin resistance-associated protein, FmtC (872 aas; 14 N-terminal TMSs (residues 1-530) plus a largely hydrophilic DUF2156 domain (residues 531-872). Similar throughout its length to FmtC of Staphylococcus aureus (2.A.1.3.35). Residues 67-326 (TMSs 2-8) are homologous to residues 527-786 (TMSs 8-14) in Rv0585c of Mycobacterium tuberculosis (2.A.1.3.43). Residues 6-314 are also homologous to an extra hydrophobic domain in Mg²⁺ P-type ATPases (3.A.3.4.3 and 3.A.3.4.4). The C-terminal domain belongs to the DUF2156 superfamily. Homologues of the hydrophilic domain retrieved with NCBI BLAST searches are annotated as "putative" lysylphosphatidyl-glycerol synthetase. Some include full length MFS fusion proteins.	Bacteria	*FmtC of Brucella melitensis* (D1F3T8)**

4.H.1.1.3	Putative MFS permease of 537 aas and 13 TMSs. TMSs 10-13 show substantial sequence similarity with TMSs 2-5 in 9.B.111.1.1 and 9.B.111.1.2).	Bacteria	*Putative MFS permease of Bilophila wadsworthia* (E5Y3Y1)**

4.H.1.1.4	Bifunctional lysylphosphatidylglycerol flippase/synthetase of 850 aas and 15 TMSs in a 14 + 1 TMS arrangement, MprF. The structure of the C-terminal synthase domain has been determined (Hebecker et al. 2015).		MprF of Bacillus licheniformis

4.H.1.1.5	Bifunctional alanyl phosphatidylglycerol flippase/synthetase MprF of 878 aas and 15 TMSs in a 14 + 1 TMS arrangement.The structure of the synthase domain has been determined (Hebecker et al. 2015). There may be a C-terminal TMS in addition to the 15 TMSs cited above.		MprF of Pseudomonas aeruginosa

4.H.1.1.6	Annotated as a putative lysyl tRNA synthetase with 7 putative N-terminal TMSs plus one putative C-terminal TMS. Pylogenetically, it clusters with the lysyl phosphatidyl glycerol synthases (ligases).	Cyanobacteria	Putative fusion protein of Anabaena variablis

4.H.1.1.7	DUF2156 domain-containing protein of 893 aas and 15 putative TMSs, 14 N-terminal and 1 C-terminal. The DUF2156 domain is the C-terminal hydrophilic domain.		DUF2156 protein of Bifidobacterium pseudolongum

4.H.1.1.8	Uncharacterized protein of 867 aas and 14 putative TMSs.		UP of Lactobacillus plantarum

4.H.1.1.9	Bifunctional lysylphosphatidylglycerol flippase/synthetase, MprF, of 848 aas and 14 TMSs in a 2 + 6 + 6 arrangement.		MprF of Hyphomonas adhaerens

Examples:
TC#	Name	Organismal Type	Example
4.H.1.2.1	Rv0585c; 795aas: 1 - 220aas, TMSs 1-6; 221-490aas, kinase domain; 491-795, TMS: 7-14. The C-terminal 8 TMS hydrophobic domain is homologous to an N-terminal domain in fused Mg²⁺-ATPases (3.A.3.4.3 and 3.A.3.4.4) and members of the MFS.	Bacteria	*Rv0585c of Mycobacterium tuberculosis* (O53781)**

4.H.1.2.2	Uncharacterized flippase-like domain-containing protein of 305 aas and 10 TMSs.		UP of Streptomyces humi

4.H.1.2.3	Flippase-like domain-containing protein of 835 aas and 16 TMSs in a 6 + 6 + 4 TMS arrangement.		Flippase of Jiangella alba

4.H.1.2.4	Uncharacterized protein of 1068 aas and 14 TMSs in a 6 + 5 + 3 TMS arrangment (UPF0104 family). .		UP of Bacillus pumilus