Bacterial protein systems at the membrane interface

Bacterial protein systems at the membrane interface

Structural and biophysical studies of the E. coli adhesion receptor intimin and the magnesium transporter MgtA

Dissertation for the degree of Ph.D.

by

Julia Anna Weikum

Centre for Molecular Medicine Norway Nordic EMBL Partnership for Molecular Medicine

University of Oslo

July 2020

© Julia Anna Weikum, 2020

Series of dissertations submitted to the

Faculty of Mathematics and Natural Sciences, University of Oslo No. 2316

ISSN 1501-7710

All rights reserved. No part of this publication may be

reproduced or transmitted, in any form or by any means, without permission.

Cover: Hanne Baadsgaard Utigard.

Print production: Reprosentralen, University of Oslo.

I Table of contents

Acknowledgments ...III List of publications ... IV Abbreviations ... V

1. Introduction ... 1

1.1 Pathogenic Escherichia coli ... 2

1.1.1 Enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli (EHEC) ... 2

1.2 Adhesion ... 3

1.2.1 Initial adherence and colonization ... 5

1.2.2 Translocation of bacterial signals into the host cell via a type three secretion system 5 1.2.3 Intimate adherence and pedestal formation of A/E lesions ... 6

1.2.4 Intimin ... 7

1.2.4.1 Structural features of intimin ... 8

1.2.4.2 Inverse autotransporter ... 9

1.2.4.3 Autotransport mechanism of inverse autotransporters ...10

1.3 E. coli cell envelope ...11

1.3.1 Glycerophospholipids ...12

1.3.1.1 Cardiolipin ...13

1.3.1.2 Adaption of lipid composition ...15

1.3.1.3 Lipid autooxidation ...15

1.3.2 Membrane proteins ...16

1.3.2.1 P-type ATPases ...17

1.3.3 Protein-lipid-interactions ...20

1.4 Magnesium-transport in E. coli ...22

1.4.1 Magnesium transporter A (MgtA) ...23

1.4.1.1 The role of cardiolipin for MgtA-mediated Mg²⁺ transport ...24

1.4.1.2 Phylogenetic distribution of MgtA ...25

II

1.4.1.3 Transcriptional and translational regulation of mgtA expression...26

1.4.1.4 Cellular functions of MgtA ...29

2. Aims of this thesis ...31

3. Synopses of publications ...33

Paper I: The extracellular juncture domains in intimin adopt a constitutively extended conformation and induce restraints in the intimin reach and sphere of action ...33

Paper II: The bacterial magnesium transporter MgtA reveals highly selective interaction with specific cardiolipin species ...34

Paper III: The Mg²⁺ sensing region of MgtA resides in the C-terminus and is dependent on pH ...35

4. Discussion ...36

5. Summary ...56

6. Future perspectives ...58

7. References...60 Annex: Paper I-III

III

Acknowledgments

The work presented in this PhD thesis was carried out at the Centre for Molecular Medicine (NCMM), University of Oslo, Norway from April 2017 to January 2019 and at DTU Bioengineering, Danish Technical University, Denmark from February 2019 to June 2020. Financial support was provided by NCMM core funding, the Research Council of Norway, NordForsk and DTU funding.

BioCat provided travel grants for courses and national conferences attended during the PhD studies.

First, I would like to thank my primary supervisor, Jens Preben Morth, who has given me the opportunity to follow my interest in protein biochemistry and pursue my PhD in his research group. I always appreciate and value his input and comments, innovative ideas and support inside as well as outside of the laboratory. I would also like to thank my co-supervisors, Ole Andreas Løchen Økstad and Reidar Lund, for their support and taking interest in my progress.

Secondly, I would like to thank all my colleagues in Oslo and Copenhagen, with whom I had the opportunity and pleasure to work with. I would especially like to thank Saranya Subramani for her contributions to my PhD thesis. Thank you for always being available when I needed guidance or support. Further, I would like to thank the former members of the Morth group, Bojana Sredic, Harmonie Perdreau-Dahl and Johannes Bauer, for the friendly welcome, great scientific input and cozy coffee breaks. I also thank Lisa Gerner for her feedback on my PhD thesis.

Additionally, I would like to thank my group members at DTU, Lisa Merklinger and Emilie Müller, for interesting discussions during lunch breaks and fun-filled activities outside of work. Thank you for making my year in Copenhagen a great time. Lastly, I would also like to thank Line Vejby Jægerum, who contributed to my project as a master’s student and remained a friend after.

I thank my office mates and co-workers at NCMM and DTU for the scientific and non- scientific discussions in the laboratory and at the coffee machine. Further, I would like to extend my thanks to the technical and administrative staff at DTU and NCMM for their support. I would like to especially thank Nina Modahl, Anita Skolem and Elisa Bjørgo, who helped me with the administrative struggles during my relocation from Oslo to Copenhagen.

I would like to thank my family for their encouragement and support in going abroad to perform my PhD studies. Further, I would like to thank my father Gerhard, my mother Liz and my sister Maria for all the valuable feedback on my PhD thesis. I thank all my friends I met in Norway, who made my time outside of the laboratory delightful, allowed me to make great memories and fall in love with the country.

Lastly, I would like to thank my partner, Magnus Wannebo, for always being there for me, cheering me up and motivating me. Thank you for being you.

IV

List of publications

This thesis is based on the following scientific articles:

I. Weikum J, Kulakova A, Tesei G, Yoshimoto S, Jægerum LV, Schütz M, Hori K, Skepö M, Harris P, Leo JC, Morth JP (2020).

The extracellular juncture domains in the intimin passenger adopt a constitutively extended conformation inducing restraints to its sphere of action.

Accepted in Scientific Reports (Nature Publishing Group).

II. Weikum J, van Dyck J, Subramani S, Klebl DP, Storflor M, Muench SP, Abel S, Sobott F, Morth JP (2020).

The bacterial magnesium transporter MgtA reveals highly selective interaction with specific cardiolipin species.

Manuscript under revision.

III. Subramani S, Weikum J, Sredic B, Perdreau-Dahl H, Langbach-Hein K, Vilsen B, Morth JP (2020).

The Mg²⁺ sensing region of MgtA resides in the C-terminus and is dependent on pH.

Manuscript in preparation.

V

Abbreviations

A/E Attaching and effacing Arp Actin-related protein

AT Autotransporter

ATP Adenosine triphosphate A-domain Actuator domain

BAM β-barrel assembly machinery Big Bacterial immunoglobulin-like BFP Bundle-forming pili

CL Cardiolipin

Cls Cardiolipin synthase

C12E8 Octaethylene glycol monododecyl ether DDM n-Dodecyl β-D-maltoside

ecMgtA E. coli magnesium transporter A E. coli Escherichia coli

EF-P Elongation factor P EHEC Enterohemorrhagic E. coli EPEC Enteropathogenic E. coli

EspFU E. coli secreted protein F in prophage U GFP Green fluorescent protein

HUS Hemolytic uremic syndrome IAT Inverse autotransporter Ig Immunoglobulin

IRD Intrinsic ribosome destabilization IRTKS Insulin receptor tyrosine kinase substrate kb kilobase

Km Michaelis-Menten constant LEE Locus of enterocyte effacement LysM Lysin motif

MC Monte Carlo

MD Molecular dynamics

VI MgtA Magnesium transporter A

MgtB Magnesium transporter B

Nck Non-catalytic region of tyrosine kinase adaptor protein 1 N-domain Nucleotide binding domain

N-WASP Neural Wiskott–Aldrich syndrome protein

kDa Kilodalton

PE Phosphatidylethanolamine PG Phosphatidylglycerol

POPE 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine P-domain Phosphorylation domain

ROS Reactive oxygen species

Salmonella Salmonella enterica serovar Typhimurium SAXS Small angle X-ray scattering

SDS-PAGE Sodium dodecyl sulphate-polyacrylamide gel electrophoresis SEC Size exclusion chromatography

Sec Secretion

SERCA Sarco/endoplasmic reticulum Ca²⁺-ATPase SP Signal peptide

stMgtA Salmonella magnesium transporter A stMgtB Salmonella magnesium transporter B T3SS Type III secretion system

Tir Translocated intimin receptor

TM Transmembrane

Tm Lipid transition temperature UPEC Uropathogenic E. coli

Vmax Maximal velocity V. cholerae Vibrio cholerae

1

1. Introduction

Escherichia coli (E. coli) is a versatile bacterial species and a common member of the human gut microbiota ¹. However, as a pathogen E. coli remains a global health threat. Certain strains, including enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli (EHEC), are among the most common causes for foodborne diarrheal diseases ², responsible for millions of acute illnesses and hundreds of deaths annually ³. A deep understanding of the bacterial infection process, ranging from host cell adhesion and colonization to the bacterial defense mechanisms against the human immunological response, is essential for the identification of novel bacterial drug targets and the development of treatments. In this thesis two E. coli proteins, which present potential drug targets, have been structurally and biophysically characterized. The first part of this thesis focused on the virulence factor intimin, essential for the bacterial adhesion process, while in the second part the bacterial magnesium transporter A (MgtA) and its regulation through lipid and magnesium interactions has been studied.

In the following, a short overview of pathogenic E coli (Section 1.1) with a focus on EPEC and EHEC (Subsection 1.1.2) will be given. As the first part of this thesis focuses on the adhesion receptor intimin, the bacterial adhesion process to the host cell (Section 1.2), including its three substages of initial adherence and colonization (Subsection 1.2.1), translocation of bacterial signals into the host cell (Subsection 1.2.2) and intimate adhesion and pedestal formation (Subsection 1.2.3), will be presented. Lastly, the virulence factor intimin will be introduced (Subsection 1.2.4). In the second part of the thesis the interaction between membrane proteins and lipids was investigated on the E. coli bacterial magnesium transporter A (MgtA) and cardiolipin. Therefore, the E. coli cell envelope (Section 1.3) and its main components, glycerophospholipids (Subsection 1.3.1) and membrane proteins (Subsection 1.3.2) with a focus on P-type ATPases (Subsection 1.3.2.1) will be shortly introduced. Additionally, an overview of the interaction between lipids and proteins will be presented (Subsection 1.3.3). Lastly, Section 1.4 will give an overview of Mg²⁺ transport in bacteria with a focus on MgtA (1.4.1).

2 1.1 Pathogenic Escherichia coli

E. coli is a Gram-negative bacterium from the family Enterobacteriaceae. Various strains of E. coli persist as commensal bacteria in the mucosa of the gastrointestinal tract ⁴. They colonize the mucus layer of the cecum and colon within hours of human birth and remain among the most abundant facultative anaerobic bacteria of the human intestinal microflora during human life ⁴. However, there are several known pathogenic E. coli strains, which have acquired virulence attributes that allow them to adapt to new environmental niches and cause a wide spectrum of diseases ¹. Common intestinal pathogens are EPEC and EHEC, which adhere to the small bowel and colon, respectively ¹. EPEC and EHEC will be described in detail below (Section 1.1.1). E. coli also plays a role as an extraintestinal pathogen. Uropathogenic E. coli (UPEC) is a common cause for urinary tract infections and meningitis-associated E. coli, which spreads through the blood circulation and translocate to the central nervous system, has been increasingly implicated with cases of meningitis and sepsis ¹. Although these pathogenic E. coli strains use different mechanisms and virulence factors during their infection process, all exhibit a multi-step infection pathway. Classical steps include adhesion and colonization of the mucosal site, multiplication and finally, emergence of disease and host damage. A major obstacle for bacterial infection is the human immune system, therefore immune evasion is inevitable for a successful infection ^1,5.

1.1.1 Enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli (EHEC)

Both, EHEC and EPEC, are among the most common foodborne pathogens and are regarded as a global health threat ^6,7. EHEC is a highly infectious bacterium, responsible for hemorrhagic colitis (bloody diarrhea) or the potentially fatal hemolytic uremic syndrome (HUS) ⁸. Symptoms of the HUS are microangiopathic hemolytic anemia, reduced levels of thrombocytes and acute renal failure ⁹. The largest EHEC outbreak occurred in 2011 in Germany with more than 850 reported cases of HUS and 54 deaths ¹⁰. EPEC, on the other hand, is a major contributor to fatal infantile diarrhea ^11,12.

Both E. coli species belong to the attaching and effacing (A/E) family of gastrointestinal bacteria. A common characteristic of this family is the formation of A/E lesions on the host cell ⁵. These are characterized by cytoskeletal rearrangements and formation of actin-rich pedestals on which the adherent bacteria reside ^2,13. Further, these lesions exhibit the disappearance of surface microvilli ¹³. The mechanism of bacterial-induced pedestal formation has been extensively

3

investigated, yet the physiological importance of the pedestal formation and subversion of the actin network is still not well understood ¹⁴. EHEC distinguishes from EPEC as its main virulence factor, the Shiga-toxin, is not present in EPEC ⁵. The toxin disrupts protein synthesis, leading to the death of intoxicated epithelial or endothelial cells ¹.

1.2 Adhesion

Bacterial infection begins in most cases with the adhesion of bacteria to the host cell. In the case of EPEC and EHEC, we distinguish three essential stages within the adhesion process, which are required for a successful adhesion event ¹⁵. These stages are shown in Figure 1. First, initial adherence and colonization of the intestine occurs (Figure 1a) ¹⁵. Upon initial adherence, bacterial signals are translocated into the host cell via the type 3 secretion system (T3SS) as the second stage of adhesion (Figure 1b). Lastly, intimate adherence is mediated by the bacterial proteins intimin and the translocated intimin receptor (Tir) (Figure 1c) ¹⁵. Upon intimate adherence, cytoskeletal rearrangements and pedestal formation of A/E lesions occur in the host cell (Figure 1d) ¹⁵. In the following, these stages of bacterial adhesion will be described in detail (Section 1.2.1 - 1.2.3).

4

Figure 1: The adhesion process of EHEC and EPEC is divided into three substages, resulting in pedestal formation of A/E lesions

The adhesion process of EHEC and EPEC can be divided into three substages: (a) Initial adherence of the bacteria to the mammalian host cell; (b) Translocation of bacterial signals into the host cell; (c) Intimate adherence of bacteria to the host cell through intimin-Tir interaction. (d) After the adhesion process cytoskeletal rearrangements occur in the host cell, leading to the pedestal formation of A/E lesions. Adapted from Kaper et al. (2004) and Croxen and Finlay (2010) ^1,5.

5 1.2.1 Initial adherence and colonization

Initial adherence requires specific adhesion proteins, which allow close interaction between the bacteria and the epithelium (Figure 1a) ¹. Whereas EPEC initially adheres to the epithelial cells in the small bowel, EHEC attaches to the epithelial cells in the colon ¹. Although EHEC and EPEC colonize different intestinal regions, they both share several common adhesion proteins ¹⁵. However, pathotype-specific adhesins have also been identified.

Among the shared adhesion proteins of EHEC and EPEC are the fimbria sorbitol- fermenting protein (Spf), type 1 fimbriae, long polar fimbria (LPF), curli and porcine A/E associated adhesin (Paa) ¹⁵. Fimbriae are rod-like structures with a large diameter of 5-10 nm that can interact with the host cell interface ¹. Although fimbriae are the most distinct morphological structures, adhesins can take several forms, including as afimbrial surface proteins ¹⁶. Adhesins commonly interact with different binding partners on the host cell. In many cases interaction with extracellular matrix proteins has been observed, for example long polar fimbria 1 and curli can interact both with fibronectin and laminin. However, curli also responds to MHC class 1 molecules ¹⁷.

An EPEC-specific adhesin is the bundle-forming pili (BFP) ¹⁵. These pili are rope-like filament bundles that mediate interbacterial adherence and support formation of bacterial networks as well as promote adherence to the host cell epithelium ¹⁵. Yet, as adhesins are essential for the initial adherence of bacteria to their host cells, the large overlap of shared adhesins indicates low contribution of adhesins for mediation of host specificity.

1.2.2 Translocation of bacterial signals into the host cell via a type three secretion system

The second step of the adhesion process involves translocation of bacterial signals into the host cell and subsequent interference with host signal transduction pathways. This step is highly dependent on T3SS, a specialized protein secretion apparatus located in the bacterial membrane, which injects effector proteins into the host ¹⁸ (Figure 1b). Genes for the T3SS, as well as regulators, chaperones and effector proteins, are encoded on a 35 kilobase (kb) chromosomally located pathogenicity island, called locus of enterocyte effacement (LEE) ¹⁴. This pathogenicity island is widely distributed among Gram-negative pathogens. Besides E. coli, it is found in Salmonella enterica serovar Typhimurium (hereafter referred to as Salmonella), Yersinia and

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Shigella ¹⁴. Although the genes encoding T3SS structural proteins are conserved between species, large variations have been detected among secreted effector proteins ¹⁴.

The T3SS is a complex machinery consisting of more than 20 proteins. It forms a syringe- shaped structure containing a central channel of 2-3 nm in diameter, which is protruding through the bacterial surface into the host cell ¹⁸. The syringe is attached to three ring structures embedded in the inner and outer bacterial membrane, which are connected through the periplasmic inner rod ¹⁸. The needle is essential for secretion of T3SS effector proteins.

Interestingly, effector proteins are unfolded when they are transported through the channel as the channel size could not accommodate folded proteins ¹⁸. Effectors secreted by T3SS exhibit a large functional variety and affect multiple cellular pathways ¹⁹. Among these are effectors that are involved in subverting innate immune pathways, including phagocytosis, inflammatory signaling pathways and regulation of host cell survival by promotion or inhibition of apoptosis ¹⁹. For a more detailed overview of bacterial effectors, refer to Santos & Finlay (2015) ¹⁹ and Pinaud et al. (2018) ²⁰. Among the injected effectors is also a receptor protein called Tir, which is essential for the third stage of adhesion, the intimate adherence of bacteria ²¹.

1.2.3 Intimate adherence and pedestal formation of A/E lesions

Tir plays an essential role for the third step of adhesion, the intimate adherence of the bacteria to the host. Upon injection into the mammalian cell by T3SS, Tir is presented outside the host cell membrane, where it binds to the virulence factor intimin on the E. coli surface (Figure 1c) ²¹. Both, intimin and Tir, are encoded on the LEE pathogenicity island ^22,23. Tir harbors the intimin binding domain, consisting of two helices separated by a hairpin loop, which is presented on the surface of the host cell ²⁴. There it interacts with the C-terminus of intimin protruding out of the bacterial outer membrane and, following, promotes the tight attachment of the bacteria to the host cell ²⁴. Intimin-Tir interaction activates actin reorganization, which induces formation of pedestal structures on the host cell ²⁵. However, EPEC and EHEC use different intracellular host mechanisms for actin pedestal formation (Figure 1d).

In EPEC, Tir recruits host cell factor neural Wiskott–Aldrich syndrome protein (N-WASP) and the actin-related protein (Arp) 2/3 complex ²⁶. These promote actin nucleation and eventually lead to actin polymerization ²⁶. It has been hypothesized that the recruitment of both host cell factors is mediated through a tyrosine residue on Tir ²⁷. Phosphorylation of the tyrosine induces Tir binding to the mammalian non-catalytic region of tyrosine kinase adaptor protein 1 (Nck). Nck

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is involved in initiation of actin signaling ²⁷. Additionally, the N-terminus of Tir directly binds to cytoskeletal protein α-actinin, mediating a stable anchor and contributing to the pedestal formation ²⁸. However, this process occurs independently of the tyrosine phosphorylation on Tir ²⁸. On the other hand, the Tir receptor specific for EHEC lacks the tyrosine residue involved in phosphorylation and does not require Nck for pedestal formation ²⁹. Tir contains an Asn-Pro- Tyr (NPY458) sequence in the C-terminus that interacts with insulin receptor tyrosine kinase substrate (IRTKS), a key regulator of membrane and actin dynamics. IRTKS recruits secreted bacterial effector EspFU (E. coli secreted protein F in prophage U), which together form a ternary complex with N-WASP ³⁰. N-WASP activates Arp2/3-complex mediated signaling processes for cytoskeletal rearrangements ³¹.

Efficient downstream signaling is further promoted through dimerization of intimin and Tir, which promotes receptor clustering ³². Additionally, different intimin variants have been shown to interact with various eukaryotic proteins, such as nucleolin or β1-integrin, which contribute to the intimate adherence of the bacteria ^33,34.

1.2.4 Intimin

Intimin, a 94 kilodaltons (kDa) surface protein, is an essential adhesin for EHEC and EPEC infection. Homologues are also found in other pathogens, such as UPEC ³⁵, Citrobacter rodentium and Hafnia alvei ³⁶. Intimin is uniformly expressed over the entire bacterial membrane ³⁷. However, intimin expression is regulated by environmental and host cell factors. Its expression levels increase during the exponential growth phase and decrease following adhesion to the host cell ³⁷. Intimin variants have shown variability in the exposed C-terminal region, allowing the generation of more than 20 variants and allele-specific subtypes of intimin. The most common clinically relevant types are the α- and β-type, found in several EPEC strains including the common strain O127:H6, and the γ-type, which is only detected in specific EPEC strains and EHEC O157:H7 ¹⁷.

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Figure 2: Intimin exhibits the classical structural composition of inverse autotransporters

(a) Schematic of the structural composition of intimin containing a periplasmic, a transmembrane and an extracellular segment.

(b) Structural comparison between an inverse and a classical autotransporter. Adapted from Leo et al.

(2015) ³⁶.

(c) Hypothesized process of intimin passenger secretion according to the hairpin model described for classical autotransporters. Adapted from Leo et al. (2012) ³⁸.

1.2.4.1 Structural features of intimin

Structurally, intimin can be divided into three segments. Intimin contains a small periplasmic sequence, including a lysin motif (LysM) domain and a signal peptide (SP), and a β-barrel located in the outer membrane ³⁶. The third segment is the extracellular region consisting of four bacterial immunoglobulin-like (Big) domains (D00-D0-D1-D2) topped by a C-type lectin-like domain (D3)

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at the C-terminus (Figure 2a) ³⁶. In the following, we will refer to the extracellular region only as the passenger, as recommended by Drobnak et al. (2015) ³⁹. The structural composition of intimin corresponds to the typical assembly of an inverse autotransporter, which will be discussed in detail further on (Section 1.2.4.2).

The periplasmic domain of intimin was shown to mediate receptor dimerization, important for receptor clustering during the adhesion process ⁴⁰. The bacterial outer membrane is spanned by the 12-stranded anti-parallel β-barrel of intimin, followed by an elongated linker that extends into the barrel pore ⁴¹. Intimin requires the β-barrel assembly machinery (BAM) complex for its proper insertion into the outer membrane ⁴², and a putative BAM signature sequence has been identified in the final strand of the β-barrel ⁴¹. The extracellular passenger consists of a chain of subdomains, which form a rod-shaped protruding extension ³⁶. However, its exact composition has long been under debate as only structural information of the C-terminal subdomains D1-D2- D3 has been obtained ²⁴. Intimin subdomains D2 and D3 form a superdomain, interacting directly with Tir ²⁴. D3 exhibits a C-type lectin-like domain, while D1 and D2 exhibit the typical immunoglobulin (Ig)-fold of Big domains. The Ig fold is typically composed of 70-100 amino acids, which are arranged in seven anti-parallel β-strands, organized in two β-sheets and packed against each other in a β-sandwich ⁴³. The β-strands are composed of alternating hydrophobic and hydrophilic residues with the hydrophobic side chains pointing towards the interior of the domain ⁴³. Different subsets of Ig-like domains have been identified and are distinguished by their topology. The canonical feature of Ig-like domains is a disulfide bridge between two conserved cysteine residues ⁴³, which is not present in intimin subdomains D1 and D2. An additional Big domain (D0) at the N-terminus of the intimin passenger has been predicted based on sequence similarity ²⁴. The presence of another subdomain, termed D00, located at the interface of the β- barrel and the extracellular passenger has been suggested, yet no structural fold could be proposed ⁴¹. In 2016, Leo et al. predicted, based on homology-based structure prediction, that D00 also exhibits an Ig fold ⁴⁴. However, no high-resolution structural information of D00 was obtained.

1.2.4.2 Inverse autotransporter

Intimin is the prototype of the type Ve secretion system, termed inverse autotransporter (IAT).

The name derives as its extracellular passenger, located at the C-terminus, is exported with the help of an N-terminally located β-barrel ³⁶. Therefore, it exhibits a reverse conformation in

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comparison to the family of classical autotransporters (AT), which are characterized as type Va secretion systems ³⁶. Figure 2b shows structural differences between the passenger of IATs and classical ATs. The passenger of classical ATs is located at the N-terminus followed by the membrane-embedded β-barrel ³⁶. Additionally, while the passenger of ATs is formed by extended β-helices, the passenger of IATs typically consists of a chain of Big domains, often capped with a C-type lectin-like domain at the C-terminus ³⁶.

Besides intimin, many other IATs have been identified, including Yersinia invasin, E. coli FdeC and Salmonella virulence factor SinH ^45,46. Especially, Yersinia pseudotuberculosis invasin has been in the focus of research as it exhibits a similar composition and function compared to intimin ^41,47. In general, IATs share several common structural features. Most prominently is the β-barrel domain and its linker region ⁴⁶ (Figure 2b). Large variations between the C-terminal passengers of IATs have been detected. Some IATs do not contain any Big domains or only a short C-terminal extension, while for others up to 47 Ig-like domains have been predicted ^45,46. Interestingly, IATs are almost exclusively found in Gammaproteobacteria ⁴⁶.

1.2.4.3 Autotransport mechanism of inverse autotransporters

Autotransporters secrete their passenger autonomously across the outer membrane, independent of adenosine triphosphate (ATP) hydrolysis or membrane potential as an energy source ³⁸. However, the exact mechanism for the secretion through the β-barrel remains unclear.

Figure 2c illustrates an initial model, adapted from classical ATs, which has been proposed for intimin passenger secretion.

In the first step unfolded intimin is exported through the cytoplasmic membrane into the periplasm via the secretion (Sec) machinery dependent on a N-terminal signal peptide (Figure 2c – Step 1) ³⁶. Periplasmic folding and outer membrane insertion of intimin is dependent on specific periplasmic chaperones (Figure 2c – Step 2) ⁴². DsbA catalyzes the formation of disulfide bonds in intimin subdomain D3 ⁴². The general chaperone SurA prevents protein aggregation and assist in the insertion of the N-terminal region into the outer membrane ⁴². Chaperones Skp and DegP play a secondary role in intimin insertion ⁴². However, for IAT invasin DegP has been linked to quality control of the insertion process ⁴⁸. Insertion of the β-barrel into the outer membrane is an essential step in the autotransport process as the passenger will be secreted through the pore into the extracellular space ⁴⁹. Yet, β-barrel insertion is dependent on auxiliary factors, such as the BAM complex ^41,42 (Figure 2c – Step 2). For the secretion of the passenger of IATs itself a

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hairpin model has been proposed. A short linker region between the β-barrel and the passenger forms a hairpin intermediate inside the membrane pore, which initiates secretion (Figure 2c – Step 3) ^36,48. Following, the passenger is pulled through the membrane pore in a way that the N- terminus of the passenger reaches the extracellular milieu first. Sequential folding of individual Big domains has been proposed as the main driving force for secretion, resulting in complete secretion and folding of the intimin passenger (Figure 2c –Step 4 & 5) ⁴⁴. Leo et al. revealed that disturbance of the Ig fold through insertion of a tag or deletion of a β-strand results in stalling of passenger secretion ⁴⁴.

However, the hairpin model has been under debate. An involvement of the BAM complex for passenger export, next to its support for β-barrel insertion, has been proposed as the β-barrel remains associated with the BAM complex despite being fully folded and inserted into the membrane ^36,44. Yet, no direct evidence for the involvement of the BAM complex in passenger secretion of IATs has been identified, but its role in classical ATs secretion has been revealed ^50,51.

1.3 E. coli cell envelope

Biological membranes are key cellular components as they separate cells from the extracellular environment and allow functional compartmentalization. They consist of a lipid matrix and embedded and attached proteins ⁵². Yet, each type of cell membrane has distinct functions dependent on the complex lipid mixture and the unique set of associated proteins ⁵². The cell envelope of Gram-negative bacteria, like E. coli, consists of four compartments: the inner membrane, the periplasm, the peptidoglycan layer, also referred to as the cell wall, and the outer membrane ⁵³. The inner, or cytoplasmic, membrane is the primary permeability barrier of the cell.

It contains specific transport proteins and permeases as well as enzymes involved in ATP and phospholipid synthesis ⁵³. The outer membrane exposes the antigenic determinants, lipopolysaccharides, to the environment and functions as a passive barrier against substrates with large molecular weights, for example antibiotics ⁵³. However, both membranes consist mainly of the same building block, glycerophospholipids ⁵³.

The classical fluid mosaic model by Singer and Nicholson has long been the standard model for describing the lipid bilayer. It characterizes the bilayer as an unperturbed, homogenous surface with random distribution of lipids and few embedded membrane proteins ⁵⁴. However, recent research has updated this model. It has been highlighted that local areas composed of

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specific lipids are present, often described as regions of functional specialization, and that the membrane bilayer is packed with intercalating membrane proteins ⁵⁴. In the following, the two main components of the bacterial membrane, glycerophospholipids (Section 1.3.1) and membrane proteins (Section 1.3.2), and their interaction with one another (Section 1.3.3) will be discussed in detail.

1.3.1 Glycerophospholipids

The most abundant lipids in E. coli membranes are glycerophospholipids ⁵⁵. The glycerophospholipid structure can be divided into two functional parts: the hydrophilic headgroup and the hydrophobic tail consisting of two long hydrocarbon chains, referred to as acyl chains (Figure 3a). The lipid backbone is a sn-3-glycerol-3-phosphate, which is esterified to the acyl chains at the first and second position ⁵². The lipid head group is linked via its phosphoryl group at position three. Chemically diverse structures can be found as headgroups and their composition defines the different lipid classes. Examples of different lipid head groups are shown in figure 3a. The acyl chains of the hydrophobic tail consist of fatty acids or fatty alcohols, varying in their length and degree of saturation ⁵⁶. In eubacteria, fatty acid chain length varies typically from 12 to 18 carbons and can be fully saturated or monounsaturated ⁵⁶. Acyl chain properties are classically described by the symbol x:y, in which x refers to the number of carbon atoms and y to the number of double bonds present in the fatty acid chains ⁵². The modular composition of lipids is possible through variations of the polar headgroup or the composition of the acyl chains, which allows a large molecular variety of glycerophospholipids ⁵⁷. The principal glycerophospholipid in the E. coli cytoplasmic membrane is the zwitterionic primary amine- containing phosphatidylethanolamine (PE), which makes up ~75-85 % of the total lipid content ^53,55. It functions primarily as a structural lipid in membranes as it forms hydrogen bonds with neighboring polar groups ⁵⁸. The remaining lipid components, phosphatidylglycerol (PG) (~10-20 %) and cardiolipin (CL) (~5-10 %), are both anionic lipids ⁵³. However, CL exhibits a unique structure with distinct features, which will be discussed in detail below (Section 1.3.1.1).

The properties of membrane bilayers are highly dependent on the physical and chemical characteristics of the lipids they contain (Figure 3b). Next to reactivity and charge of the polar headgroup, the composition of the hydrophobic tail also plays a major role ⁵⁷. Acyl chain composition regulates membrane viscosity as lipids with saturated acyl chains pack with higher density and tend to form non-fluid gel phases ⁵⁷. They are characterized by a high degree of acyl

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chain order, reduced lateral lipid mobility and increased membrane viscosity ⁵⁷. However, monounsaturated acyl chains exhibit a kinked shape, leading to less tight packing and increased mobility. Therefore, they tend to form fluid bilayers at physiological temperatures ⁵⁷. Polyunsaturated lipids differ in their physical properties from both saturated and unsaturated lipids ⁵⁷. However, bacteria lack polyunsaturated lipids ⁵⁹. Lipid properties are also sensitive to temperature. The lipid transition temperature (Tm) is defined as the temperature, at which lipids transition from the ordered gel phase, which contains closely packed lipids, to the liquid disordered phase, in which acyl chains are randomly oriented and show increased fluidity ⁶⁰.

1.3.1.1 Cardiolipin

Cardiolipin (1,3-diphosphatidyl-sn-glycerol) is a unique phospholipid dimer. As a phospholipid dimer, its major structural difference in comparison to classical glycerophospholipids is that a single headgroup glycerol is shared by two phosphatidate moieties ⁶¹. Therefore, the CL structure essentially consists of two glycerophospholipid molecules with four acyl chains in total, as shown in figure 3a. As both phosphodiester moieties should be negatively charged under physiologically relevant conditions, CL is classified as an anionic lipid ⁶¹. Additionally, CL has a very small headgroup in comparison to its hydrophobic tail. The small head size enhances the probability of CL to form inverted non-lamellar lipid phases and its presence in the lipid bilayer induces negative curvature stress ⁶¹. The cell poles of E. coli and other bacteria are often enriched in CL ⁶². Based on the conical lipid shape of CL, it was proposed that CL clusters at the cell poles due to spontaneous, transverse and lateral lipid microphase separation ⁶³. However, the physical properties of CL-containing membranes depend on various factors including acyl chain composition, the solvent environment, the presence of counter cations and the composition of the remaining lipid environment ⁶³. Although the percentage of CL normally remains low in the bacterial membrane, it is strongly involved in the cellular energy metabolism through interactions with enzymes involved in oxidative phosphorylation and ATP synthesis ⁶⁴. Due to its negative charge and organization of microdomains, a function of CL as a proton sink has been proposed ⁶⁴. Further, a role of CL as a “flexible linker” has been suggested, filling cavities at protein interfaces and allowing stabilization of individual subunits of oligomeric complexes ⁶⁴. It was shown that the mobility of the CL headgroup is impaired, leading to reduced intra- and intermolecular interactions with other headgroup moieties. Therefore, the steric self-hindrance of CL in the lipid bilayer is likely reduced, which allows a higher accessibility of CL phosphate groups to interact with water,

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metal ions and integral or surface bound membrane proteins ⁶¹. This highlights the specific significance of CL for lipid-protein interactions.

As CL contains four acyl chains, more than 100 different CL species are theoretically possible through different combinations of acyl chains varying in their saturation degree and length. However, an unexpected symmetry in lengths of the four acyl chains of CL has been observed in biological membranes ⁵⁹. It was proposed that an imbalance between chain lengths may disrupt the CL resonance structure and four acyl chains of the same length will symmetrically stabilize the CL headgroup ⁵⁹. In E. coli, 56 species were identified with palmitic acid (16:0), palmitoleic acid (16:1) and oleic acid (18:1) as the most abundant acyl chains ⁶⁵. Three enzymes are responsible for CL synthesis in E. coli. Cardiolipin synthase A (ClsA) synthesizes CL in the exponential phase, while cardiolipin synthase B and C (ClsB and ClsC) contribute to the lipid synthesis in the stationary growth phase ⁶⁶.

In eukaryotes CL is essentially only found in the mitochondrial membrane, where it contributes up to ~20 % to the total lipid content ⁶⁷. Despite the large molecular diversity and number of potential CL species, CL is often limited to one or two major species in eukaryotic cells, which account for 60-90 % of the total CL fatty acid mass ⁶⁸. As an example, CL extracted from bovine heart contains linoleic acid (18:2) as the dominant acyl chain ⁶⁹.

Figure 3: Glycerophospholipids are a major building block of biological membranes

(a) Schematic of the composition of classical glycerophospholipids in comparison to the phospholipid dimer cardiolipin.

(b) Physicochemical properties of the lipid bilayer dependent on lipid composition. Adapted from Ernst et al. (2016) ⁵⁷.

15 1.3.1.2 Adaption of lipid composition

The lipid composition of bacterial cells is highly adaptable to changes in the environment.

Adaption is mediated through variations of the lipid class or the acyl chain composition of bacterial membranes ⁵⁷. During the bacterial growth cycle the proportion of CL can increase from ~5 % up to ~30 % in the stationary phase ⁶⁵, which has been linked to increased CL synthase activity ⁷⁰. Additionally, changes in osmolarity lead to increased proportions of anionic phospholipids and decreased zwitterionic lipids ⁷¹. Especially, the proportion of CL has been observed to increase under osmotic stress or impaired energy metabolism ⁷¹. The function of CL under osmotic stress is not known. However, it was proposed that an upregulation of CL synthesis allows CL- dependent regulation of osmosensory transporter ProP ⁷¹.

Additionally, fatty acid composition in E. coli adapts to environmental conditions. A classical phenomenon, homeoviscous adaptation, describes the adaptive response of the lipid environment to changing temperatures in order to maintain membrane fluidity ⁵⁷. While the polar head group composition remains unchanged, increased percentages of saturated acyl chains are detected at elevated temperatures ^72,73. Further, elevated temperatures also exhibit an increase in cyclopropane fatty acid formation ⁷⁴. Cyclopropane increase also occurs under high salt conditions ⁷⁵, acidic media and growth under anaerobic conditions ⁷⁶. Additionally, the acyl chain composition also changes depending on the bacterial growth phases with decreasing levels of unsaturated fatty acids as the cultures progresses from the exponential growth phase to the stationary phase ⁷⁷.

1.3.1.3 Lipid autooxidation

As lipids are essential for cell compartmentalization, damages to the lipid integrity can destabilize membranes, thus increasing membrane permeability and leading to cell lysis and death ⁷⁸. A common cause for destruction of membrane integrity is lipid autooxidation ⁷⁹. This process occurs through a radical chain mechanism upon interaction of lipids with reactive oxygen species (ROS).

Lipid autooxidation is a self-propagating and self-accelerating process and it is divided in three distinct stages: initiation, propagation and termination ⁷⁹. During initiation, unsaturated lipids lose a hydrogen atom and form free radicals in the presence of initiators, such as light, heat or metal ions. In the second stage, propagation, the lipid radical reacts with oxygen and forms a peroxyl radical, which can attack a new lipid molecule and induce a rapidly progressing reaction. This

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reaction will be repeated until no hydrogen source is available anymore or when the propagation reaction is interrupted, for example by antioxidants ⁷⁹. This represents the final stage, termination, and concludes the lipid autooxidation process. Although a variety of lipid oxidation products have been identified, hydroperoxides represent the primary products ⁸⁰. However, they can decompose further into various oxygenated and aliphatic fatty acid scission products ⁸⁰. Lipid stability depends on various factors, including the degree of unsaturation of fatty acids, making highly unsaturated lipids most susceptible to oxidation ⁸⁰. Lipid oxidation has been shown to influence the integrity of biological systems altering membrane fluidity and membrane composition and disrupting lipid- protein interactions ^78,81.

1.3.2 Membrane proteins

The membrane bilayer is packed with membrane proteins involved in various cellular functions ranging from transporters moving molecules across the membrane to receptors transmitting signals between the intra- and extracellular environment. Like lipids, membrane proteins are amphipathic. They can be classified into two general categories: integral (intrinsic) and peripheral (extrinsic) proteins ⁵². Integral proteins are tightly bound to the membrane through hydrophobic forces and they contain one or more segment that is embedded in the membrane ⁵². In most cases integral proteins even span the entire phospholipid bilayer, therefore being classified as transmembrane proteins. Peripheral proteins are not embedded into the lipid bilayer but only attach to the membrane surface. They do not interact with the hydrophobic core of the lipid bilayer, but rather with the surface exposing domains of lipid molecules or integral proteins ⁵².

As membranes promote cellular compartmentalization, a major function of membrane proteins is maintaining ion homeostasis across the membrane. Proteins that transport ions across the lipid bilayer are separated into two classes: ion channels and ion pumps ⁸². The first class, ion channels, are passive conduits. Ions rush through channels dependent on concentration and electric potential gradients ⁸². The second class, ion pumps, require energy, released from ATP hydrolysis or another source, to actively transport ions against gradients ⁸². Therefore, they play an essential role in building up ion gradients and electric potentials across membranes ⁸². A major difference between ion channels and pumps is that the first class only requires the presence of a single gate, which restricts ion movement along the translocation pathways. An ion pump, on the other hand, needs at least two gates, which should never be open at the same time to avoid uncontrolled ion movement ⁸².

17 1.3.2.1 P-type ATPases

The P-type ATPase family constitutes a large class of ion transporters ⁸³. P-type ATPases are involved in establishing and maintaining steep electrochemical gradients across biological membranes, making them essential for all eukaryotic organisms and most prokaryotes ⁸⁴. P-type ATPase- mediated ion transport is coupled to ATP hydrolysis, which provides the energy required for ion transport across the respective membranes ⁸³. A wide variety of ions are being transported by P-type ATPases as substrates ⁸³. Among the most studied and well-known representatives of this transporter family is the Na⁺-K⁺-ATPase, responsible for maintaining the electrochemical gradient across the cytoplasmic membrane in most animal cells ⁸³. In the focus of research has also been the sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA), which pumps Ca²⁺ back into the lumen of the sarcoplasmic reticulum during muscle relaxation ⁸³. Further, the gastric H⁺-K⁺- ATPase, which maintains low pH in the stomach lumen by active transport of H⁺, has been extensively investigated ⁸³. Lastly, plasma membrane H⁺-ATPases are essential for the establishment of membrane potentials in plants and fungi, which energize the plasma membrane for cellular processes ⁸⁵. There are five branches of P-type ATPases with more than ten subgroups, which are mainly distinguished by their transported ions (Appendix A1) ^84,86,87.

1.3.2.1.1 Structural features of P-type ATPases

All P-type ATPases are multi-domain membrane proteins with molecular masses between 70-150 kDa ⁸⁸. P-type ATPases share a common structural composition, which is illustrated in figure 4a.

The main catalytic unit (α-subunit) consists of six to twelve transmembrane (TM) α-helices and comprises the ion transport domain ⁸⁴. In some cases this domain is divided into two subdomains, called T-domain, consisting of the first six, and S-domain, consisting of the remaining TM- helices ⁸⁷ (Figure 4a). While the T-domain harbors the ion binding sites and is present in all P- type ATPases, the S-domain is an auxiliary unit, which can have specialized functions, such as ion-coordination or additional ion binding sites ⁸⁷. Both, the C- and N-terminus of P-type ATPases, are on the membrane side facing the cytoplasm, therefore, all P-type ATPases have an even number of TM segments ⁸⁸.

Additionally, all P-type ATPases have three cytoplasmic domains, which confer the ATP- hydrolyzing activity ⁸⁴. These are termed the N-domain (nucleotide binding domain), P-domain (phosphorylation domain) and A-domain (actuator domain) ⁸⁴ (Figure 4a). The N-domain performs

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ATP binding and phosphorylates the P-domain ⁸⁷. During this process, the N-domain recognizes and positions the γ-phosphoryl of ATP for a nucleophilic attack ⁸⁴. Additionally, coordination with Mg²⁺ allows to bring the ATP molecule closer to the phosphorylation site and promotes the nucleophilic attack and phosphoryl transfer ⁸⁹. All P-type ATPases contain a highly conserved aspartate residue in the P-domain, which accepts the phosphoryl group and forms a high energy aspartyl-phosphate intermediate ⁸⁴. The A-domain functions as a built-in phosphatase involved in dephosphorylation of the P-domain. A glutamate residue in the A-domain positions a water molecule for the subsequent hydrolysis and release of the phosphoryl group ⁸⁴.

Essential for ion transport is the conversion of the energy released through ATP hydrolysis in the cytoplasm to the physical translocation of ions through the TM segment ⁸⁴. This is conversed through five linker regions that connect the cytoplasmic domains to the transmembrane section.

It was revealed that the integrity and proper length of these linkers is highly relevant for P-type ATPase activity. The deletion of single residues in the linker region severely affected the catalytic activity of SERCA, even leading to complete inhibition ^90,91.

The ion binding sites are located in the TM domain between helices M4, M5, M6 and M8, where they are coordinated by negatively charged and polar residues ⁸⁹. Interestingly, SERCA, the Na⁺-K⁺- and the H⁺-K⁺-ATPase show high similarity in their ion binding sites, although they transport different ions in different quantities in each direction ⁸⁹. This indicates that the same binding sites reorient to accommodate different ions. Yet, distribution and number of charged amino acids in the sites likely have been adapted to the respective ions ⁸⁹. Additionally, it proposes that ion selectivity might rather be mediated through a gating mechanism and a selectivity filter at the ion entrance pathway ⁸⁴.

1.3.2.1.2 Catalytic cycle of P-type ATPases

To achieve active transport of ions against an electrochemical gradient, an open passage across the membrane needs to be avoided as this would result in rapid flow-back of the transported ions ⁸⁹. P-type ATPases use an alternate-access model, in which both membrane sides are transiently closed and transported ions are occluded ⁸⁹. Selected transport is then mediated through opening of a narrow pathway to either membrane side. This transport mechanism is only possible through extensive conformational changes of P-type ATPases, driven by ATP hydrolysis ⁸⁹. The catalytic cycle follows the Post-Albers scheme and alternates between several

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different conformational states. Two main conformations, termed E1 and E2 state, are distinguished, which differ in their affinity towards the transported ions ⁸⁷ (Figure 4b).

The first state, E1, exhibits high affinity for ion 1, which is the ion transported out of the cytoplasm ⁸⁹. Ion binding triggers phosphorylation of the P-domain by Mg²⁺-ATP, forming the high- energy E1P·ADP state. Release of ADP induces conformational changes and the transporter reaches the low-energy state E2P ^88,89. These conformational changes distort the ion binding site(s) and lower the affinity for ion 1. Simultaneous opening at the extracytoplasmic side results in exit of ion 1 to the extracytoplasmic space ^88,89. Conformational state E2P binds ion 2, referred to as the counterion, with high affinity. Binding of ion 2 induces re-occlusion and dephosphorylation, resulting in conformational switching from the E2P to the E2 state. The release of the counterions into the cytoplasm completes the cycle and induces transition to the E1 state again ^88,89. Exchange of ions occurs through half-channels oriented towards the cytoplasmic side (E1-ATP state) or the extracytoplasmic side (E2-P state) ⁸⁷.

Figure 4: P-type ATPases are a large family of ion transporters moving ions across membranes according to the Post-Albers scheme

(a) P-type ATPases classically consist of a transmembrane and a cytoplasmic segment with an actuator (A-), a phosphorylation (P-) and a nucleotide (N-) domain.

(b) Schematic overview of the P-type ATPase transport cycle according to the Post-Albers scheme. Blue refers to E1 states with high affinity for ion 1 (light blue); Red refers to E2 states with high affinity for ion 2/

counterion (orange). Adapted from Palmgren and Nissen (2011) ⁸⁷.

20 1.3.3 Protein-lipid-interactions

In the past the lipid bilayer was mostly considered to function only as a solvent media for membrane proteins ⁵⁴. However today, the importance of lipid-protein interactions has been highlighted for hundreds of membrane proteins ⁹² and the characterization of lipid-protein interactions has allowed a better understanding of cell membrane organization. Lipids have been implicated with various membrane protein functions. These range from protein activity, stability and oligomerization as well as protein localization, folding and topology ^92–94.

Lipid-protein interplay can occur through interactions between selected lipid molecules binding to a specific site on the protein or through the general physicochemical properties of the lipid bilayer dependent on its composition ⁹³. Figure 5 shows the three classical categories of lipid- protein interactions: bulk lipids, annular lipids and non-annular lipids ⁹³. Bulk lipids are lipid molecules present in the bilayer, which interact non-specifically with the membrane protein. They exhibit fast diffusion rates and low resilience time at the protein surface ⁹³. The second category, annular lipids, are comprised of lipids from a selected class or molecular species. They often interact with either hydrophobic or hydrophilic membrane protein surfaces ⁹³. Simulations indicated that a membrane protein is typically engulfed by 50-100 lipid molecules that form a shell, termed annular lipid shell, around the TM segment ⁹⁵. These lipids exhibit a reduced exchange rate in comparison to bulk lipids ⁹³. The last category, non-annular lipids, are specific lipid molecules that often bind at selective site-specific binding sites on the protein. These lipids often reside within the membrane protein complex. They exhibit a low exchange rate and, due to the tight interaction, can in many cases be co-purified with membrane proteins ⁹³.

Additionally, physicochemical properties of the lipid bilayer can also affect membrane protein functions ⁹³. The hydrophobic thickness of the bilayer, typically between 35-55 Å, is defined by its lipid composition. Hydrophobic mismatch describes the impaired compatibility between the hydrophobic surfaces of the membrane protein to the lipid bilayer ⁹³. In case of incompatibility, local changes occur through recruitment of additional lipid molecules, deformation of the membrane or conformational changes of the protein. Hydrophobic mismatch has been revealed as a molecular mechanism for transporter regulation ⁹³. Additionally, lateral pressure, which is defined as the pressure exerted by the membrane on its components located inside it, has been shown to affect membrane proteins. The highest pressure is typically at the interfacial region between hydrophobic and hydrophilic areas. However, lateral pressure is also dependent on local asymmetries across the bilayer, the lipid order and hydrophobic mismatch ⁹³. In the

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following, selected examples of lipid-protein interactions and their role for protein functions will be presented.

The importance of the lipid environment or specific lipid interactions for protein activity has been observed for many membrane proteins, including several representatives of the P-type ATPase family. The ATPase activity of SERCA, including its affinity for Ca²⁺, is highly dependent on the membrane thickness and fluidity of the surrounding bilayer ^96,97. SERCA requires a mismatch between hydrophobic thickness of the bilayer and its membrane embedded part for optimal flexibility ⁹⁸. Norimatsu et al. (2017) performed an extensive study on the first layer of phospholipids of SERCA crystal structures in four different conformational states ⁹⁹. They proposed that the lipid environment plays a more active role for SERCA function and is directly involved in the dynamics of its pump function. Local distortions of the lipid bilayer occur through TM helices movement, which can be used as an energy source contributing to conformational changes. Additionally, modulation of catalytic activity mediated through the lipid environment has been shown for several other members of the P-type ATPase family, including Cu(I)-transporter CopA and heavy metal transporter ZntA ^100–102.

Lipids have also been linked to protein stabilization. The high prevalence of membrane protein crystal structures with bound lipids has revealed a stabilizing effect early on ¹⁰³. Today, native mass spectrometry (MS) has become a useful tool to analyse specific lipid-protein interactions and several cases of lipid-mediated stabilization of protein oligomers have been revealed ^104,105. For the Na⁺-K⁺-ATPase, a specific lipid site was identified that only affected protein stabilization, but showed no effect on protein activity ¹⁰⁶. A second lipid binding site only affecting protein activity was also characterized, but both effects are independently modulated by different lipid classes.

Further, membrane protein topology has been shown to not only be determined by the amino acid sequence, but it can also be influenced by the lipid composition of the membrane.

Most membrane proteins follow the positive-inside rule, in which they orient their positively charged amino acid residues facing the cytoplasm ¹⁰⁷. Negatively charged phospholipids have been shown to inhibit translocation of positively charged domains, contributing to membrane protein topology ¹⁰⁸. The topology of several E. coli transporter, containing multiple TM helices, was severely affected depending on the lipid composition of the membrane. The presence of PE has been shown to be required for the correct orientation of the transporters lactose permease LacY and γ-aminobutyric acid permease GabP ^109,110. Additionally, membrane protein assembly was affected upon changes of membrane fluidity ¹¹¹.

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Lastly, lipids have also been implicated in protein localization. Many proteins have been identified at the bacterial cell poles, where they co-localize with CL ¹¹². Therefore, it has been hypothesised that CL is involved in the polar recruitment or positioning of certain membrane proteins, including osmosensory protein ProP ¹¹³. However, as the mechanism and the cellular machinery for polar localization of lipids and proteins is not completely understood yet, showing direct effects of lipids on protein localization remains an obstacle.

Figure 5: Lipids can affect membrane protein function through different types of interaction Side view (a) or top view (b) of a membrane protein engulfed by the membrane bilayer, exhibiting different types of lipid interactions, which vary in their exchange rate. Adapted from Contreras et al. (2013) and Stangl and Schneider (2015) ^93,114.

1.4 Magnesium-transport in E. coli

Magnesium is the most abundant divalent cation in cells and contributes to many cellular processes ¹¹⁵. These range from stabilization of macromolecules, including ribosomes and membranes, to neutralization of nucleic acids and nucleotides and a function as a co-factor in many enzymatic processes ¹¹⁵. Recently, Mg²⁺ homeostasis has also been shown to be important for bacterial survival and defense against the host innate immune response. Magnesium deprivation has been revealed to be the main resistance mechanism of host resistance factor

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SLC11A1 against Salmonella infection in macrophages ¹¹⁶. However, the chemical properties of Mg²⁺ differ greatly from other cations. As an example, the ionic radius of Mg²⁺ is among the smallest of all cations (0.65 Å) while its hydrated radius is more than 400 times larger ¹¹⁷. In comparison, the hydrated radius of Ca²⁺ and K⁺ is only 25 or four times larger than their dehydrated from, respectively ¹¹⁷. This highlights the complex requirements of Mg²⁺ transporters in terms of recognition of the largely hydrated ion, removal of the hydration shell and transport of the bare ion ¹¹⁷.

Bacteria contain three distinct transporter classes for magnesium uptake: CorA, magnesium transporter E (MgtE) and bacterial magnesium transporter A and B (MgtA/MgtB) ¹¹⁵. Many bacterial species express multiple Mg²⁺ transporters of the same or different classes ¹¹⁵. The constitutively expressed transporters CorA or MgtE exhibit a large phylogenetic distribution and are considered the primary Mg²⁺ transporters ¹¹⁵. Both use the electrochemical gradient across the cytoplasmic membrane as their energy source for Mg²⁺ transport, essentially mediating influx and efflux of Mg²⁺¹¹⁵. Therefore, they are considered channels rather than transporters ¹¹⁵. On the contrary, MgtA and its homologue MgtB only mediate Mg²⁺ influx ¹¹⁸. Initial research revealed that MgtA/MgtB mediate Mg²⁺ uptake only if cells are present in an environment containing low Mg²⁺ concentrations and expression of these transporters has been shown to be dependent on extracellular Mg²⁺ levels ¹¹⁹. In recent years a tight and complicated regulatory network of MgtA and MgtB expression and activation has been identified, which will be discussed in detail below (Section 1.4.1.3). Most of our understanding of bacterial Mg²⁺ homeostasis derives from the Gram-negative bacterium Salmonella ¹¹⁵, containing Mg²⁺ transporter CorA, MgtA and MgtB ¹¹⁹. However, large similarities between E. coli, which contains Mg²⁺ transporter CorA and MgtA, and Salmonella Mg²⁺ homeostasis have been described ^117,120.

1.4.1 Magnesium transporter A (MgtA)

E. coli MgtA (ecMgtA) consists of 898 amino acids and has a molecular weight of 99.5 kDa ¹²¹. It is located in the bacterial inner membrane ¹²² and as a P-type ATPase its C- and N-terminus points into the cytoplasm. MgtA belongs to the PIIIB subfamily of P-type ATPases, closely related to the yeast and plant proton transporters of the PIIIA subfamily ⁸⁴. Further, MgtA is phylogenetically closer to eukaryotic P-type ATPases, for example SERCA, than prokaryotic ATPases ¹²³. As a P- type ATPase, MgtA utilizes ATP hydrolysis for the Mg²⁺ transport from the periplasm to the cytoplasm. Interestingly, no direct evidence for Mg²⁺ transport by MgtA has been obtained yet.