Outer membrane sorting and assembly pathway

The outer membranes of mitochondria, chloroplasts and Gram-negative bacteria contain β- barrel proteins, a special type of integral membrane proteins. A beta barrel is a large beta- sheet that twists and coils to form a closed structure in which the first strand is hydrogen bonded to the last207. So far identified β-barrel proteins are porin (or VDAC), Tom40, Tob55

(Sam50), Mdm10 and Mmm2. Like all other mitochondrial outer membrane proteins, they are synthesized in the cytosol. The β-barrel precursors are imported by the TOM complex and inserted into the outer membrane by a translocase named TOB (topogenesis of mitochondrial outer membrane β-barrel) or SAM (sorting and assembly machinery) (Figure 2.2)208,209. Precursors of β-barrel proteins interact mainly with the receptor Tom20 of the TOM complex and are subsequently transported through the translocase to the trans side of the outer membrane, where TIM chaperone complexes guide the hydrophobic precursors to the TOB complex210,211. Its main component is the essential Tob55 (Sam50), which is homologous to the bacterial outer membrane protein Omp85208,212. Several partner proteins of Tob55, namely Tob38 (Sam35), Mas37 (Sam37) and Mdm10 (Mitochondrial distribution and morphology 10), aid in membrane insertion and assembly of the β-barrel precursors. The exact function of Tob38 and Mas37 is not yet known. Mdm10 together with the two other morphology proteins, Mdm12 and Mmm1 (Maintenance of mitochondrial morphology 1) forms a complex that functions in the assembly pathway of β-barrel proteins downstream of the TOB complex213_. The N-terminal hydrophilic region is exposed to the IMS and forms a characteristic structure, called POTRA (polypeptide translocation associated) domain214. The POTRA domain of Tob55 may be involved in the recognition of β-barrel precursors and might pass them on to the membrane-embedded C-terminal region of Tob55, which then facilitates membrane insertion and assembly of the β-barrel proteins171,215.

2.2.3

The TOM complex

The molecular machine translocating proteins across the mitochondrial outer membrane is the TOM complex (translocase of the outer mitochondrial membrane, Figure 2.2). This large multisubunit membrane protein is responsible for the initial recognition of precursor proteins in the cytosol and subsequent transfer of the polypeptides through pores. It also facilitates release of cytosolic-binding factors and contributes to the unfolding of cytosolic protein domains. The holo-TOM complex is composed of at least seven different subunits. Tom20 and Tom70 are the major receptors that recognize preproteins, whereas the subunits Tom40, 22, 7, 6 and 5 form the stable TOM core complex. Tom20 and Tom70 are both anchored with N-terminal transmembrane segments in the outer membrane and expose hydrophilic domains into the cytosol. They differ in their substrate specificity, but overlap in their function and can partially substitute each other. As shown in the crystal structure of Tom70216 _{it contains} several conserved tetratricopeptide repeat (TPR) motifs, which are organized in a right-

handed superhelix. These motifs form a peptide-binding groove for specific interaction with cytosolic chaperones like Hsp70, whereas the C-terminal part of Tom70 reveals a putative binding site for precursor proteins. Tom70 shows substrate preference for hydrophobic precursors that comprise internal targeting signals216,217_{. In contrast, Tom20 is the central} receptor for N-terminal presequences and structural analysis by NMR demonstrated a binding groove for the hydrophobic face of the MTS (matrix targeting signal, see Section 2.2.2.1)218. The binding sites for precursor proteins on the cytosolic site are often referred to as cis- binding sites, whereas the IMS-exposed binding surface of the TOM complex represents the

trans-binding site171.

Tom40 represents the pore-forming component of the complex and was proposed, based on theoretical predictions, to adopt a β-barrel fold (see Section 2.2.2.4). Even in the absence of other TOM subunits, purified Tom40 forms pores in artificial membranes that show characteristics to that of the entire TOM complex219-221. Tom22, Tom5, Tom6, and Tom7 each have a single α-helical transmembrane segment that locks them tightly into position on Tom40222. Tom22 spans the outer membrane in a Nout-Cin orientation exposing a highly negatively charged N-terminal domain to the cytosol and a short C-terminal part to the IMS. It assists the transfer of substrate proteins from the receptors to the pore and may cooperate with Tom20 in the binding and unfolding of precursor proteins. In addition, Tom22 plays a critical role for the general integrity of the TOM complex171,223-226. The small TOM proteins (Tom5, Tom6 and Tom7) appear to function in regulating the stability of interactions within the complex, thereby assisting substrate protein transfer to and through the core translocase. The loss of individual small TOM subunits causes only minor effects, but simultaneous deletion of all three is lethal227-229_{. Mutational analysis in Neurospora crassa revealed that Tom6 plays a} major role in TOM complex stability, whereas Tom7 has a lesser role229. The TOM holo complex (purified in the detergent digitonin) has a molecular mass of roughly 490-600 kDA and single-particle imaging of the negatively stained TOM holo complex showed particles with two or three pore-like structures, whereas the TOM core complex, which lacks Tom20 and Tom70, contains two pores with a diameter of ~2.1 nm230-232. Isolated Tom40 alone is capable of forming homooligomeric structures, which reveal one cavity220,221,233. Further studies by electron microscopy report that Tom20 is selectively responsible for the presence of the three pore-like structure. Both subunits Tom22 and Tom20 appear to be critical for the assembly of Tom40 channel units232.

In addition to translocation of precursor proteins, the TOM complex is also involved in the integration of outer membrane proteins into the membrane, a process which is only partially understood171,234. The structural organization of the TOM machinery is still a subject of

debate and three-dimensional structures will shed light on its overall topology and import mechanism.

2.2.4

Goals of this study

In the outer membrane of mitochondria the TOM complex represents the main entry site for distinct types of mitochondrial precursor proteins. The complex interaction modes of receptor subunits with preproteins and subsequent translocation of unfolded substrate proteins through the membranes are actively investigated, but still very poorly understood. The filamentous fungi Neurospora crassa turned out to be an excellent model organism for studying the TOM complex due to its fast growth rate and simple manipulation procedures. The aim of this work was the improvement of crystals of the TOM core complex to get initial structural information of the translocase complex. Therefore, the purification strategy of TOM core complex had to be optimized and the crystal quality should be improved by additive screening and crystal manipulation approaches as well as by detergent exchange. Since crystallization in complex with antibody fragments could facilitate the crystallization and structure determination of membrane proteins, one major goal of this work was to derive TOM-directed monoclonal antibody fragments from classical hybridoma technology to accomplish amelioration of the diffraction quality by co-crystallization. Another aim of this study was the recombinant expression of the main TOM component Tom40 in E. coli and its subsequent refolding to obtain highly pure protein suitable for crystallization.

2.3

Results