1
Supplementary Results & Discussion – Pulldown experiments:
2
Böck and coworkers were the first ones to isolate intermediate Hyp-complexes by
3
means of StrepTactin-affinity chromatography (Maier et al. 1998). They demonstrated that
4
tagged E. coli HypC- or HypD-homologues were able to bind and coelute with their
5
associated complex partners upon affinity chromatography. Similar experiments have been
6
employed to isolate or visualize other putative catalytic intermediates, and to characterize
7
these complexes biochemically (Bürstel et al. 2012; Chan et al. 2012; Stripp et al. 2013). The
8
major difficulty faced with studies in the native organisms is the presence of untagged native
9
constituents of the endogenous [NiFe]-hydrogenase maturation machinery. In heterologous
10
hosts, conditions for complex formation are readily controlled by selecting tagged and
11
putatively associated genes to be coexpressed.
12
In order to isolate putative complexes between maturation factors, plasmids encoding
13
genes for the production of StrepII-tagged baits with putative binding partners were generated
14
and expressed in E. coli (Supplementary Table I). In most cases, low-temperature
15
autoinduction strategies were required to yield soluble gene products, which proved to be
16
even more critical for the AH-derived M2-homologues. The isolated complexes were
17
analyzed by SDS-PAGE in order to determine their composition.
18
HypC is a small chaperone and plays a central role in Fe-cluster insertion. It is known
19
to interact directly with HoxH (Jones et al. 2004). Further, HypC forms a complex with
20
HypD, which coordinates the iron-group and receives the cyanide ligands, a process that
21
involves additional docking of cyanylated HypE (see Fig. 1A) (Bürstel et al. 2012; Stripp et
22
al. 2013; Watanabe et al. 2007). Using C-terminally StrepII-tagged variants of HypC1 or its
23
homologue from the AH set, complexes with HoxH were readily detected (Supplementary
24
Fig. 1A,E). Notably, the comparable weak pulldown of HoxH using HypC1 (M1), and the
25
stronger association with HypC2 (M2) supports the previous notion that a stronger binding of
26
non-native HypC-homologues interferes with subsequent steps of maturation, thereby causing
27
reduced processing efficiencies of non-native intermediates (see main body of the article).
28
The
tagged
HypC
baits
also
formed
complexes
with
HypD-homologues
29
(Supplementary Fig. 1B,F,I). Notably, these complexes were intact, yet inactive in cell-free
30
maturation assays when purified aerobically. The integrity of the corresponding isolates was
31
greatly improved by moving cell-opening and purification steps into an anaerobic glove box.
32
In all cases, elution fractions contained the tagged bait in stoichiometric excess. Fe-cluster
33
assembly and insertion necessitates interactions between HypCD-complexes and cyanylated
34
HypE. In the case of E. coli Hyp-proteins, a stoichiometric HypCDE-complex has previously
35
been isolated (Blokesch 2004). The complexes isolated in this work yielded two dominant
36
species at about 90 and 180 kDa by native PAGE and size-exclusion chromatography, which
37
correspond to monomeric and dimeric forms of the (HypCDE)n-complex (Supplementary Fig.
38
1C and Supplementary Fig. 3). Notably, HypE1 did not coelute with HypC1D1 (M1) unless
39
the hypF gene was coexpressed with hypE. This is in line with the proposal for the E. coli
40
HypCDE-complex that HypE-homologues can only enter a preformed HypCD-complex in
41
their cyanylated (“modified”) form, an event that is crucially preceded by HypF-mediated
42
transcarbamoylation (Blokesch 2004). Interestingly, pulldown of AH-derived HypC2D2E2
43
yielded only trace levels of the complex (Supplementary Fig. 1G), suggesting that the stability
44
of the latter is strongly reduced.
45
Apparently, HypE and HypF do not form a stable complex, as judged by pulldown
46
experiments using either StrepII-tagged HypE- or HypF-variants as baits for coelution of the
47
respective binding partner, which altogether failed to produce isolatable complexes. The
48
HypEF-complex has so far only been detected in an environment containing all other
49
components of the maturation sequence (Blokesch et al. 2004; Jones et al. 2004), or by
50
mixing the two purified proteins after their separate production and isolation (Shomura and
51
Higuchi 2012). Arguably, the complex is transiently formed but subsequently dissociates after
52
completion of the transcarbamoylation-dehydration reactions. In the absence of a preformed
53
Fe-HypCD-complex, HypE-S-C≡N (C-terminally cyanylated HypE) is likely to reside in the
54
cytosol as a dead-end intermediate.
55
For studies of the nickel-insertion complexes, HypB-homologues were used as the
56
tagged baits, which led to the isolation of intact HypAB-complexes of both sets
57
(Supplementary Fig. 1D,H). We further tested, if interactions between parts of the SH- and
58
the AH-set could be stably isolated. This was successful for a stoichiometric complex
59
between StrepII-tagged HypC1 (M1-set) and HypD2 (M2-set) (Fig. 6I). Apart from this
60
interaction, neither the coproduction of tagged baits from the SH-specific set in cells
61
expressing the AH-maturases, nor the production of baits from the latter set in cells producing
62
the former proteins yielded cross reacting material (data not shown).
63
HypX (which is encoded in the MBH-operon of C. necator; Fig. 1B), tagged either C-
64
or N-terminally, was prone to aggregation under all conditions tested. Purification of the
65
soluble protein was not achieved, although trace levels were present in elution fractions (data
66
not shown). Since we found the solubility of many Hyp-proteins to be largely increased by
67
producing them alongside associated complex partners (e. g. HypD-homologues), this might
68
be a requirement for HypX as well. However, the role of HypX in maturation of oxygen-
69
tolerant hydrogenases is currently unknown, as are associated complex partners of the
70
maturase. Studies aiming at the analyses of these assumed interactions are currently on the
71
way.
72
73
74
75
76
Supplementary Results & Discussion – References:
77
Blokesch M. 2004. [NiFe]-Hydrogenasen von Escherichia coli: Funktionen der am
78
Metalleinbau beteiligten Proteine [PhD Thesis]: LMU München.
79
Blokesch M, Paschos A, Bauer A, Reissmann S, Drapal N, Böck A. 2004. Analysis of the
80
transcarbamoylation-dehydration reaction catalyzed by the hydrogenase maturation
81
proteins HypF and HypE. Eur J Biochem 271(16):3428-36.
82
Bürstel I, Siebert E, Winter G, Hummel P, Zebger I, Friedrich B, Lenz O. 2012. A universal
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scaffold for synthesis of the Fe(CN)2(CO) moiety of [NiFe]-hydrogenase. J Biol Chem
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287(46):38845-53.
85
Chan KH, Lee KM, Wong KB. 2012. Interaction between hydrogenase maturation factors
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HypA and HypB is required for [NiFe]-hydrogenase maturation. PLoS One
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7(2):e32592.
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Jones AK, Lenz O, Strack A, Buhrke T, Friedrich B. 2004. NiFe hydrogenase active site
89
biosynthesis: identification of Hyp protein complexes in Ralstonia eutropha.
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Biochemistry 43(42):13467-77.
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Maier T, Drapal N, Thanbichler M, Böck A. 1998. Strep-tag II affinity purification: an
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approach to study intermediates of metalloenzyme biosynthesis. Anal Biochem
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259(1):68-73.
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Shomura Y, Higuchi Y. 2012. Structural Basis for the Reaction Mechanism of S-
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Carbamoylation of HypE by HypF in the Maturation of [NiFe]-Hydrogenases. J Biol
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Chem 287(34):28409-19.
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Stripp ST, Soboh B, Lindenstrauss U, Braussemann M, Herzberg M, Nies DH, Sawers RG,
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Heberle J. 2013. HypD Is the Scaffold Protein for Fe-(CN)2CO Cofactor Assembly in
99
[NiFe]-Hydrogenase Maturation. Biochemistry 52(19):3289-96.
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Watanabe S, Matsumi R, Arai T, Atomi H, Imanaka T, Miki K. 2007. Crystal structures of
101
[NiFe] hydrogenase maturation proteins HypC, HypD, and HypE: insights into
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cyanation reaction by thiol redox signaling. Mol Cell 27(1):29-40.
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