Solution structure of the NDH-1 complex subunit CupS from
Thermosynechococcus elongatus
☆
Annika Korste
a, Hannes Wulfhorst
b, Takahisa Ikegami
c, Marc M. Nowaczyk
b, Raphael Stoll
a,⁎
a
Biomolecular Spectroscopy, Faculty of Chemistry and Biochemistry, Ruhr University of Bochum, Bochum, Germany
bDepartment of Plant Biochemistry, Faculty of Biology, Ruhr University of Bochum, Bochum, Germany c
Institute for Protein Research, Osaka University, Japan
a b s t r a c t
a r t i c l e i n f o
Article history:
Received 18 December 2014 Received in revised form 30 April 2015 Accepted 6 May 2015
Available online 14 May 2015 Keywords:
Bioenergetics/electron transfer complex CupS
Cyanobacteria Membrane proteins NDH-1 complex NMR protein structure
The cyanobacterial multi-subunit membrane protein complex NDH-1 is both structurally and functionally related to Complex I of eubacteria and mitochondria. In addition to functions in respiration and cyclic electron transfer around photosystem I (PSI), the cyanobacterial NDH-1 complex is involved in a unique mechanism for inorganic carbon concentration. Although the crystal structures of the similar respiratory Complex I from Thermus thermophilus and Escherichia coli are known, atomic structural information is not available for the cyanobacterial NDH-1 complex yet. In particular, the structures of those subunits that are not homologous to Complex I will help to understand their distinct functions. The 15.7 kDa protein CupS is a small soluble subunit of the complex variant NDH-1MS, which is thought to play a role in CO2conversion.
Here, we present the NMR structure of CupS from Thermosynechococcus elongatus, which is the veryfirst struc-ture of a specific cyanobacterial NDH-1 complex subunit. CupS shares a structural similarity with members of the Fasciclin protein superfamily. The structural comparison to Fasciclin type proteins based on known NMR structures and protein sequences of human TGFBIp, MPB70 from Mycobacterium bovis, and Fdp from Rhodobacter sphaeroides, together with a virtual docking model of CupS and NdhF3, providefirst insight into the specific bind-ing of CupS to the NDH-1MS complex at atomic resolution.
© 2015 Elsevier B.V. All rights reserved.
The cyanobacterial NDH-1 complex is located in the thylakoid mem-brane and contributes to respiration, cyclic electron transfer (CET) around PSI, and is involved in a unique mechanism for the concentra-tion of inorganic carbon. Structurally, it is closely related to the Complex I of eubacteria (e.g. Escherichia coli) and the mitochondrial respiratory chain. Despite of progress in X-ray structural analysis that revealed the structures of the membrane domain of Complex I of E. coli and the entire complex of Thermus thermophilus[13,14] structural details of cyanobacterial NDH-1 remain elusive. The active complex of E. coli comprises 14 subunits (NuoA–NuoN), of which 11 are conserved in cyanobacterial NDH-1 (NdhA–NdhK). Cyanobacteria are devoid of homologues for the three subunits responsible for NADH oxidation in eubacteria (NuoE-G) and thus require a different electron transfer path-way, presumably via ferredoxin. In addition, cyanobacterial NDH-1 con-tains the four subunits NdhL–NdhO. They are found in organisms that perform oxygenic photosynthesis and are therefore termed the
oxygenic photosynthesis-specific (OPS) domain of unknown func-tion[1]. Recently, three additional subunits have been discovered, which have been termed NdhP, NdhQ, and NdhS[6,7]. Based on re-verse genetics and proteomic studies[3,4], four different NDH-1 sub-types (NDH-1L, NDH-1L′, NDH-1MS, NDH-1MS′) with distinct functions have been postulated, of which three (1L, NDH-1MS, NDH-1MS′) have been identified on the protein level. All sub-types share the subunits that constitute the core complex NDH-1M (NdhA–C, NdhE, NdhG–K, NdhL–O, NdhS) but differ in the presence of complex-specific subunits. NDH-1L is formed by the additional subunits NdhD1, NdhF1, NdhP, and NdhQ[36,39,40], NDH-1L′ by NdhD2, NdhF1, and NDH-1MS by NdhD3, NdhF3, CupA, and CupS as well as the NDH-1MS′ complex by NdhD4, NdhF4, and CupB. All NDH-1 subtypes have been shown to be involved in CET[5], whereas NDH-1L (as well as NDH-1L′) plays a role in respiration, NDH-1MS and NDH-1MS′ participate in the unique carbon concentration sys-tem in an unknown manner (for a review see[1]). NDH-1MS′ with its specific subunit CupB is important for the constitutive CO2uptake,
while NDH-1MS with its specific subunits CupA and CupS is involved in high-affinity CO2uptake at low CO2levels[8]. The physiological
role of CupS, however, still remains elusive, since no changes in phenotype could be observed for a deletion mutant of Synechococcus sp. PCC7002[9]. The CupS protein contains 133 amino acids in
Biochimica et Biophysica Acta 1847 (2015) 1212–1219
Abbreviations: NDH-1, NAD(P)H dehydrogenase type 1; NDH-CET, NDH-1 mediated cy-clic electron transport; OPS, oxygenic photosynthesis-specific.
☆ In memoriam Prof. Dr. William S. Sheldrick, PhD.
⁎ Corresponding author at: Biomolecular Spectroscopy, Faculty of Chemistry and Biochemistry, Ruhr University of Bochum, Universitätsstraße 150, 44801 Bochum, Germany. Tel.: +49 234 32 25466.
E-mail addresses:[email protected],[email protected](R. Stoll).
http://dx.doi.org/10.1016/j.bbabio.2015.05.003 0005-2728/© 2015 Elsevier B.V. All rights reserved.
Contents lists available atScienceDirect
Biochimica et Biophysica Acta
j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / b b a b i o
Synechocystis sp. PCC 6803 (Sll1734, Uniprot: P73392) and 149 amino acids in Thermosynechococcus elongatus (Tll0220, Uniprot: Q8DMA1). It exhibits a sequence similarity to the bacterial secreted protein MPB70[2]and other members of the Fasciclin superfamily of proteins, which are known to be involved in cell adhesion.
In order to gain insight into the detailed functions and reaction mechanisms of the different NDH-1 complexes, structural information is essential. Electron microscopy studies revealed an L-shaped structure for the NDH-1L complex and a U-shaped structure for the NDH-1MS complex[10,11]. The difference is caused by the additional CupA and CupS proteins in NDH-1MS. Although the CupS subunit was too small to be distinguished precisely, it could still be located next to CupA by the use of an YFP-fusion system in combination with single particle analysis[12]. Similarly, the position and orientation of the single trans-membrane subunit NdhP in NDH-1L could recently be revealed[36]. The crystal structures of the partially homologous respiratory Complex I from E. coli [13] and T. thermophilus [14] shed light on the cyanobacterial NDH-1 complex structure. Nevertheless, in order to un-derstand the various functions of the cyanobacterial NDH-1 complexes, structural data on the NDH-1 complexes themselves are vital. In partic-ular, structures of subunits that are not homologous to any respiratory Complex I subunits are of great interest.
Here, we present the NMR solution structure of CupS, a small, 15.7 kDa subunit of the NDH-1MS complex from T. elongatus. This is the veryfirst structure of a cyanobacterial NDH-1 complex subunit and it might help to completely understand its role in the carbon uptake system in the future. Furthermore, we note the sequential and structural similarity to proteins of the Fasciclin superfamily. A comparison with the protein sequences and NMR structures of human TGFBIp[15], MPB70 from Mycobacterium tuberculosis[16], Fdp from Rhodobacter sphaeroides[17], and other Fasciclin domain proteins from eukaryotes, reveals structural features, which may be of importance for the function of CupS in the NDH-1 complex. Moreover, sequence alignments and in silico docking models suggest the putative location of CupS in the NDH-1MS complex together with its interaction partners.
1. Experimental procedures 1.1. Protein purification
The generation of the expression vector for CupS with a C-terminal Strep-tag, the overexpression of15N/13C-labelled protein,
and the purification of CupS-Strep has been reported recently[18]. The purity of CupS-Strep was judged from 12.5% polyacrylamide gel according to[37].
1.2. NMR spectroscopy
The NMR experiments were performed on a sample containing 0.5 mM protein in 50 mM Tris–HCl (pH 8.0), 50 mM NaCl, 10 mM deuterated dithiothreitol (DTT), and 10% D2O. All NMR spectra
were acquired at 298 K on BrukerBioSpin Avance-III 950, Avance-I 800, DRX-600, and DRX-500 spectrometers. Using a combination of various spectra, an almost complete backbone and side chain chem-ical shift assignment was recently obtained[18]. For structural dis-tance restraints to be used during structure calculation,15N-edited
NOESY as well as13C-edited NOESY spectra (each with a mixing
time of 100 ms) were recorded. Most of the NMR experiments in-volved WATERGATE and water-flip-back methods for suppression of the water signal, except for13C-edited NOESY spectra, which
were measured using samples dissolved in D2O-based buffer. Spectra
were processed using NMRPipe[19]. The resolution in the15N/13C
indirect dimensions of the triple-resonance experiments was dou-bled by linear-prediction, respectively. A cosine-square window function was applied to all the dimensions before Fourier transformation.
1.3. Structure calculation
Spectra were analysed and chemical shifts were assigned using CcpNmr 2.3[20]. NOEs were picked manually and obvious intraresidual and sequential NOEs were assigned. Dihedral angles were obtained from TALOS +[21], which employs Cα and Cβ chemical shift values. In addition, dihedral angles of Lys107 were added during structural re-finement to minimise the variation of these values and to improve the quality of the Ramachandran statistics. For structure calculation, ARIA 2.3[22]/CNS 1.2.1[23]and UNIO (ATNOS/CANDID)[24]/CYANA 3.0. [25]software packages were used. Both programme packages assign the picked peaks automatically during structure calculation. Intra-residual and sequential NOEs were removed for afirst calculation with UNIO/CYANA, which resulted in a well-defined structural fold. The assigned output peak lists were used for further calculations in ARIA with torsion angle dynamics, supplemented with manually picked peaks. This greatly helped to identify the folded core of CupS. After a several iterations of structural refinement, amino acids 130–159 were removed as they appeared disordered in the ensemble. Finally, 100 structures were calculated during the last iteration and the 40 best structures were refined in water, of which the 10 lowest-energy struc-tures were validated using the iCING validation package[26]. The coor-dinates of CupS have been deposited to the Protein Data Bank (www. rcsb.org) under PDB-ID accession number 2MXA.
Fig. 1. Purification of CupS-Strep. A: Purification via StrepTactin affinity chromatography. B: Further purification through size exclusion chromatography revealed a retention time for CupS-Strep of approx. 12 min.
2. Results
2.1. Protein purification
A total of 4.2 mg CupS-Strep was purified from a soluble E. coli extract of 4 l of culture using StrepTactin affinity chromatography (Fig. 1A), followed by size-exclusion chromatography (Fig. 1B).
CupS-Strep contains only one tryptophan and three tyrosine residues, leading to a molar extinction coefficient of 9970 M−1cm−1. Therefore, the
ab-sorbance of the elution peak of CupS-Strep appears to be rather small. As judged from SDS-PAGE (Fig. 2), the preparation of CupS-Strep yields pure protein.
2.2. Solution structure of CupS
Thefinal structural ensemble of CupS was calculated based on the
1H,13C, and15N chemical shift assignments (Fig. 3) as well as on 2089
distance restraints (929 intra-residual, 448 sequential, 281 medium-range and 431 long-medium-range) derived from the15N-edited (Fig. 4) and 13C-edited NOESY spectra. Moreover, 221 dihedral angle restraints
were used, which were derived from TALOS+[21]. The amino acid res-idues 130–159 were omitted from the structure calculation, because they showed only intra-residual NOEs and did not converge during structure calculation. Thefinal structure family exhibits very good RMSD (root-mean-square deviation) values and Ramachandran statis-tics as indicated by the iCING validation tool[26](seeTable 1).
The protein CupS consists of seven shortα-helices (α1 4–10, α2 16–24, α3 26–31, α4 43–51, α5 53–59, α6 62–70, α7 79–85) and sevenβ-strands (β1 36–41, β2 72–74, β3 87–90, β4 93–100, β5 104–106, β6 112–117, β7 122–126). Therefore, the 129 N-terminal
Fig. 2. SDS-PAGE monitoring of the CupS-Strep purification. A: CupS-Strep elution of StrepTactin affinity chromatography. M: marker; sol.: soluble E. coli fraction; elu: elution. B: Purity of CupS-Strep after SEC. 1 to 20μg protein was applied onto the gel.
Fig. 3. Strip plot from a 3D HNCACB spectrum of CupS that shows the sequential connec-tivity for amino acids Y77 to D81.
Fig. 4. Strip plot from a 3D15
N-edited NOESY spectrum of CupS that displays the sequen-tial NOE-based connectivity for amino acids E119 to I123 in addition to medium- and long-range NOEs.
residues of CupS adopt a rather complex fold. All structural elements are solvent-exposed, because none of them is completely buried in a hydro-phobic core. Moreover, CupS does not possess any cysteine residues and therefore any disulphide bonds. The illustration inFig. 5emphasises that theα-helical regions cluster at the one side of the protein (with the exception of helixα7), while the β-sheets cluster at the other side. Allβ-strands except for β1 and β7 are antiparallel to each other. The β1–β2–β6–β7 sheet is orthogonally oriented to the β3–β4–β5 sheet. The three N-terminal helicesα1, α2, and α3 are loosely packed against α6 and β2. The strands β1 and β2 are connected through the three α-helicesα4, α5, and α6. Furthermore, strands β2 and β3 are connected through a rather long loop that contains helixα7. The α1–α2–α3 and α4–α5–α6 segments are each arranged in a U-shaped manner. The structure family of CupS (Fig. 6) is of high precision with RMSD values as low as 0.72 ± 0.18 Å for all backbone and 1.04 ± 0.18 Å for all heavy atoms. Theα-helical region exhibits some structural variability among the members of the structure family. Especially helixα4 displays a high conformational variability (refer to comparison ofFig. 5A and B). Moreover, the exact length of helixα3 cannot be determined in all structures whereas theβ-sheet region is very well-defined, apart from strandβ2 which is more pronounced in some structures than in others. 3. Discussion
3.1. CupS and related proteins
The NMR solution structure of CupS from T. elongatus comprises seven shortα-helices and seven β-strands and is thus fairly complex.
The protein consists of 149 amino acids (Tll0220, Uniprot: Q8DMA1), 20 of which have been omitted in the structure calculation, because they neither showed long-range nor medium-range NOEs, presumably due to dynamical disorder. The same observation was made for the protein MPB70 from Mycobacterium bovis, which shows sequence sim-ilarity to CupS. Interestingly, the homologous CupS protein from Synechocystis sp. PCC 6803 (Sll1734, Uniprot: P73392) contains only 133 amino acids. Therefore, the C-terminal part either does not seem to be important for the CupS function in general or it is important for the thermophilic T. elongatus only. The sequence identity between the two proteins is 68% as calculated by PSI-BLAST (http://blast.ncbi.nlm. nih.gov).
The CupS protein is part of the NDH-1MS complex, which is located in the thylakoid membrane of cyanobacteria[1]. Nevertheless, CupS shows high sequence similarity to the Fasciclin superfamily as deter-mined by PSI-BLAST. Fasciclins are often extracellular proteins, which are involved in cell adhesion. Among them are Fasciclin I (Drosophila melanogaster), the human transforming growth factor-beta-induced protein (TGFBIp, also known asβig-h3), MPB70 (M. bovis), and the Fasciclin I domain protein (Fdp, R. sphaeroides)[29,38,17].Table 2lists the corresponding results from the DALI server and each sequence iden-tity to CupS from T. elongatus[28]. Fasciclin I is an insect cell adhesion molecule, which contains four homologous domains of approx. 150 res-idues, termed Fas1 domains 1 to 4. Attempts to crystallise full-length Fasciclin I have so far been unsuccessful, but the crystal structure of Fas1 domains 3–4 could be solved[29]. According to PSI-BLAST the se-quence identity is rather low for both the Fas1 domain 3 (22%) and the Fas1 domain 4 (14%). However, this is not surprising, because other Fas1 domains exhibit a sequence identity even lower than 20% [29]. Yet, this was the best match obtained from the DALI search (see Table 2). TGFBIp also contains four Fas1 domains. This extracellular pro-tein is found in several human tissues, especially in the cornea[30]. In-terestingly, several mutations found in TGFBIp are associated with corneal dystrophy. For the Fas1–4 domain of TGFBIp, the NMR structure
Table 1
NMR statistics of the CupS structural ensemble based on the 10 lowest energy structures from a total of 40 after water refinement in ARIA 2.3.
Distance restraints Total 2089 Intra-residual 929 Sequential 448 Medium-range 281 Long-range 431
Restraints per residue 16.2
Long-range restraints per residue 3.3
Dihedral angle restraints
φ, ψ 221
RMSD
Backbone 0.72 ± 0.18 Å
Heavy atoms 1.04 ± 0.18 Å
Ramachandran statistics
Most favoured region (%) 83.6
Additionally allowed region (%) 15.3
Generously allowed region (%) 0.5
Disallowed region (%) 0.6
N
N
Fig. 5. Ribbon representation of the NMR solution structure of CupS. The amino terminus is labelled with N. A: The energetically most favoured structure (0 NOE violationsN 0.5 Å, 2 dihedral violationsN 5°). B: The structure with the lowest violation values (0 NOE violations N 0.5 Å, 1 dihedral violations N 5°).
Fig. 6. The structure family of CupS. The stereo superposition of the protein backbone is shown in blue.α-Helical regions are depicted in red, β-sheets in green. The amino termi-nus is labelled with N.
was determined recently[15]. NMR structures are also available for MPB70 (a protein secreted from M. bovis, which causes tuberculosis in animals and might bind to cell surface proteins of the host organism) [16]as well as for Fdp (another protein with cell adhesion properties) from R. sphaeroides[17].
3.2. Structural comparison with the Fasciclin superfamily
The CupS sequences of T. elongatus and Synechocystis sp. PCC 6803 were aligned to the Fas1–4 domain of human TGFBIp, MPB70 from M. bovis, and Fdp from R. sphaeroides (Fig. 7). Fasciclin I, however, was not included due to the low sequence identity. Two highly conserved re-gions H1 and H2 of unknown function were reported previously[31] and can be identified in the alignment.
The crystal structure of the Fas1–3 domain of Fasciclin I, the NMR structures of the Fas1–4 domain of TGFBIp, MPB70, and Fdp all show a homologous core fold, which consists of sevenβ-strands and five to eightα-helices. The β-sheet region of structures from different Fasciclin superfamily members is very similar. However, theα-helical regions are structurally less conserved. The NMR structure of CupS that was deter-mined in this work exhibits a topology very close to that of the Fas1-4 domain of TGFBIp (Fig. 8, A and B). Three N-terminal helices are follow-ed by the strandβ1 and another three α-helices. A rather short β-strand β2, which is oriented antiparallel to β1, connects these helices with the seventhα-helix that precedes the rest of the rather conserved β-sheet arrangement that had been described previously. The Fas1 domain 3 of Fasciclin I only showsfive α-helices: two at the N-terminus, two be-tween strandsβ1 and β2, and one between strands β2 and β3. MPB70 possesses eightα-helices, five of which are located at the N-terminus and the remaining three are found between strandsβ1 and β2. In con-trast to the other structures described here, MPB70 does not contain an α-helix between strands β2 and β3. Fdp does not comprise this helix ei-ther, but contains a helical turn at this position.
Despite of similar topology, there is a major structural difference be-tween CupS and all other proteins described here, which is the position of the three N-terminalα-helices. In CupS, these are oriented such that they point away from the protein core to display residues of the conserved regions H1 and H2 on the surface and to expose them to the solvent (Fig. 8, C). The other protein structures, e.g. TGFBIp, have the N-terminal helices docked onto this surface (Fig. 8, D). Therefore, they are not capable to directly interact with any other proteins or ligands. Due to the distinct arrangements of these helices in comparison
to the other Fasciclin superfamily members, CupS has a different protein surface and is thus likely to function in another way. Interestingly, the three N-terminal helices and especially the residues involved in binding to the protein core in TGFBIp do not show enough variation to explain the discrepancies between the three-dimensional structures. Because of this, the 3D NOESY spectra were explicitly searched for NOEs be-tween the N-terminal residues and the surface formed by H1 and H2, but none could be found. This structural difference is presumably linked to a different mode of action, because CupS is a subunit of a membrane protein complex and therefore not likely to function in the same way as extracellular cell adhesion proteins do.
For the cell adhesion function of TGFBIp via integrin, an Asp-Ile se-quence close to the H1 and H2 regions was shown to be important [32]. These amino acids are also conserved in CupS (Asp115–Ile116), but in Fasciclin I and Fdp the isoleucine residue is substituted by a valine residue. Because these residues are buried to create an interface with the N-terminal helices (in all described protein structures except for CupS), it has been speculated that ligand binding, e.g. interaction with integrin, competes with the intramolecular binding of the N-terminal helices in an autoinhibitory mechanism[17]. This is further supported by the fact that the multiple mutations of TGFBIp (also known as βig-h3) that cause corneal dystrophy are located in the protein core, which is not accessible with the N-terminus being attached[16]. Al-though the distinct function of CupS remains elusive to date, as a part of an intracellular membrane protein complex in cyanobacteria it is de finite-ly not involved in cell adhesion and does not share the same ligands with Fdp, TGFBIp or related proteins. Thus, the autoinhibitory function of the N-terminal helices might indeed not be crucial for the function of CupS.
3.3. Binding of CupS to the NDH-1MS complex
In a previous study CupS could be located at the tip of the membrane domain of NDH-1MS next to the CupA subunit[12]. Therefore, it seems likely that CupA is an interaction partner for CupS. Together, they form the soluble part of the domain that is specific for the NDH-1MS complex. In contrast to that, the soluble part of the specific domain of NDH-1MS′ only comprises the CupB subunit, which shares a high degree of se-quence similarity with CupA (Fig. 9). However, an additional domain that encompasses amino acids 82 to 118 (Fig. 9) is unique to CupA that might constitute a potential interface for an interaction between CupA and CupS. Additionally, the localisation of CupS within the most distal part of the NDH-1MS complex suggest an interaction with the transmembrane subunit NdhF3. Based on the crystal structure of Com-plex I from T. thermophilus (PDB-ID: 4HEA) NdhF3 from T. elongatus was modelled using PHYRE2 (http://www.sbg.bio.ic.ac.uk/servers/ phyre2)[33] and screened for potential CupS docking sites using ClusPro (http://cluspro.bu.edu) [34,35]. One reasonable model is shown inFig. 10. CupS might interact through binding of the positively charged part of its N-terminal helices and a negatively charged domain including large sections of the H1 and H2 regions to a conversely shaped region with inverted electrostatic surface potential at the cytoplasmic
Table 2
PSI-BLAST and DALI results for CupS from T. elongatus.
Protein Sequence identity Z-score RMSD
FasI 1–3 22% 7.6 2.1 Å
FasI 1–4 14% 7.5 3.2 Å
TGFBIp 36% 6.6 3.3 Å
MPB70 38% 7.1 2.8 Å
Fdp 41% 5.9 2.7 Å
Fig. 7. Sequence alignment of Fasciclin superfamily members: CupS from Thermosynechococcus elongatus, CupS from Synechocystis sp. PCC 6803, the Fas1-4 domain of human TGFBIp, the secreted protein MPB70 from Mycobacterium bovis, and Fdp from Rhodobacter sphaeroides. The conserved regions H1 and H2 (according to Kawamoto et al.[31]are highlighted in blue). Residues conserved among all aligned sequences are shown in red.
side of NdhF3. The aforementioned conserved amino acids Asp115– Ile116 of CupS, which could be of functional significance, are surface-exposed and point to the proximal part of the cytoplasmic surface of NdhF3, at which the other potential binding partner CupA might be lo-cated. It is important to note that for NdhF1 and NdhF4– the respective homologues of the NDH-1L and the NDH-1MS′ complex – similar docking sites could not be identified. This underlines the selective bind-ing of CupS to the NDH-1MS complex. A tentative location of CupS with-in the NDH-1MS complex and the differently composed NDH-1MS′ is schematically shown inFig. 11. Mutational interaction studies with CupA and/or NdhF3 will help to locate the exact interaction surface be-tween these subunits and CupS. They will also contribute to the under-standing of the structural and functional differences of CupS compared to the other members of the Fasciclin superfamily.
In conclusion, we present the NMR solution structure of the cyanobacterial NDH-1MS complex subunit CupS. It consists of seven α-helices and seven β-strands. The α-helical regions cluster at the one
side of the protein (with the exception of helixα7), while the β-strands cluster at the other side. Allβ-sheets except for β1 and β7 are anti-parallel to each other. Theβ1–β2–β6–β7 sheet is orthogonal to theβ3–β4–β5 sheet. The three N-terminal helices α1, α2, and α3 are loosely packed againstα6 and β2. The strands β1 and β2 are connected by the threeα-helices α4, α5, and α6. Furthermore, strands β2 and β3 are connected through a rather long loop that contains helixα7. The α1–α2–α3 and α4–α5–α6 segments are each arranged in a U-shaped manner. CupS is sequentially and structurally similar to the Fasciclin su-perfamily. However, these proteins are usually located extracellularly, while CupS is part of the intracellular membrane complex NDH-1MS. The structure in solution reveals a major structural difference to similar proteins such as TGFBIp, MPB70 and Fdp: the N-terminal helices are not docked onto but rather point away from the protein core of CupS. Pre-sumably, CupS utilises a binding interface with the NDH-1MS complex that most likely differs from those known for established members of the Fasciclin superfamily of proteins.
Fig. 8. Structural comparison of CupS and the Fas1-4 domain of TGFBIp. The topology of both proteins is very similar (A: CupS, B: TGFBIp[15]), but the NMR structures of CupS (C) and Fas1-4 of TGFBIp (D, PDB-ID: 2LTB) show different surfaces. Regions H1 and H2 are coloured blue.
Fig. 9. Sequence alignment of CupA and CupB from Thermosynechococcus elongatus. Despite their overall homology, CupA contains an additional domain (outlined in red), presumably forming a CupS interaction site.
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Acknowledgement
Part of this work was performed under the Cooperative Research Program of the Institute for Protein Research, Osaka University, with a lot of help from members of the Prof Genji Kurisu's group in IPR. This work was supported by the RUB Research School (DFG GSC 98/3) to H.W. and by a grant of the Deutsche Forschungsgemeinschaft DFG to M.M.N. (NO 836/1-1).
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