Infinite Kinetic Stability against Dissociation of Supramolecular Protein Complexes through Donor Strand Complementation

Puorger, Chasper; Eidam, O.; Capitani, Guido; Erilov, Denis A.; Grütter, Markus G.; Glockshuber, Rudi

doi:10.1016/j.str.2008.01.013

Cited by 72 publications

(114 citation statements)

References 42 publications

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“…The typical free energy of protein folding varies between Ϫ20 and Ϫ60 kJ/mol (39). Strikingly, FimG, a structurally related pilus subunit, is stabilizes by ϳ70 kJ/mol when complemented with the FimF donor strand (40). Similar results were obtained for self-complemented FimA.…”

Section: Fimata-li-ssra)supporting

confidence: 75%

Both ATPase Domains of ClpA Are Critical for Processing of Stable Protein Structures

Kress

Mutschler²,

Weber‐Ban

2009

Journal of Biological Chemistry

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ClpA is a ring-shaped hexameric chaperone that binds to both ends of the protease ClpP and catalyzes the ATP-dependent unfolding and translocation of substrate proteins through its central pore into the ClpP cylinder. Here we study the relevance of ATP hydrolysis in the two ATPase domains of ClpA. We designed ClpA Walker B variants lacking ATPase activity in the first (D1) or the second ATPase domain (D2) without impairing ATP binding. We found that the two ATPase domains of ClpA operate independently even in the presence of the protease ClpP or the adaptor protein ClpS. Notably, ATP hydrolysis in the first ATPase module is sufficient to process a small, single domain protein of low stability. Substrate proteins of moderate local stability were efficiently processed when D1 was inactivated. However, ATP hydrolysis in both domains was required for efficiently processing substrates of high local stability. Furthermore, we provide evidence for the ClpS-dependent directional translocation of N-end rule substrates from the N to C terminus and propose a mechanistic model for substrate handover from the adaptor protein to the chaperone.The chaperone ClpA is a member of the AAA ϩ protein family (ATPase-associated with various cellular activities) and catalyzes the energy-dependent degradation of proteins through interaction with the protease ClpP in Escherichia coli (1-3). Like many other AAA proteins (4), ClpA oligomerizes into a ring structure, shaping a central pore through which substrate proteins are routed into the proteolytic core ClpP. This process involves unfolding and translocation of the substrate protein and requires the consumption of ATP. AAA proteins can be grouped into class I (two ATPase domains) and class II (one ATPase domain) ATPases. The fact that some ATPases have only one AAA module whereas others seem to require two ATPase domains stimulated researchers to investigate the roles and interdependence of the two ATP-binding modules in class I AAA proteins like Hsp104 and ClpB (5-8). ClpA also features two ATPase domains, termed D1 and D2. They are highly homologous, but differences in the amino acid sequence around the conserved regions in D1 and D2 suggested that they might have a somewhat different function (9). As was shown for several other class I members, ClpA assembles into its oligomeric state only upon binding of nucleotide (10). Indeed, substituting the invariant lysine in the Walker A motif demonstrated that nucleotide binding to D1 triggers ClpA hexamerization, whereas ATP turnover is mainly catalyzed by D2 (11, 12). However, mutations in the Walker A motif also abolish or drastically decrease the affinity for ATP, making it impossible to distinguish between effects due to ATP binding and those due to ATP hydrolysis.To study the role of ATP hydrolysis in both ATPase domains uncoupled from nucleotide binding events, we designed ClpA Walker B variants that lack the ability to hydrolyze ATP in either D1 (ClpAE286A), D2 (ClpAE565A), or both domains (ClpAE286A/E565A) but still bind ATP in bot...

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Section: Fimata-li-ssra)supporting

confidence: 75%

Both ATPase Domains of ClpA Are Critical for Processing of Stable Protein Structures

Kress

Mutschler²,

Weber‐Ban

2009

Journal of Biological Chemistry

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“…4A, lanes 3-7, and Fig. S5) and VP16 fl (lanes [8][9][10][11][12][13][14][15][16][17][18][19][20]. Whereas, HCF-1 C sequences are more important for VP16 fl than VP16 ΔAD VIC formation (Fig.…”

Section: Resultsmentioning

confidence: 90%

“…Although the interaction between two proteins through "strand complementation" has been observed in other proteins (19), the SAS1 structure was unexpected because it had been hypothesized that the SAS1C region alone could form two Fn3 domains and that these would provide a dynamic surface for reversible interaction with SAS1N making it possible to regulate HCF-1 N and HCF-1 C subunit association (15). Given the evident stability of SAS1N-SAS1C structure-as evidenced, for example, by its resistance to numerous different alanine substitutions (Fig.…”

Section: Discussionmentioning

confidence: 99%

HCF-1 self-association via an interdigitated Fn3 structure facilitates transcriptional regulatory complex formation

Park

Lammers

Herr

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Host-cell factor 1 (HCF-1) is an unusual transcriptional regulator that undergoes a process of proteolytic maturation to generate N-(HCF-1 N ) and C-(HCF-1 C ) terminal subunits noncovalently associated via self-association sequence elements. Here, we present the crystal structure of the self-association sequence 1 (SAS1) including the adjacent C-terminal HCF-1 nuclear localization signal (NLS). SAS1 elements from each of the HCF-1 N and HCF-1 C subunits form an interdigitated fibronectin type 3 (Fn3) tandem repeat structure. We show that the C-terminal NLS recruited by the interdigitated SAS1 structure is required for effective formation of a transcriptional regulatory complex: the herpes simplex virus VP16-induced complex. Thus, HCF-1 N -HCF-1 C association via an integrated Fn3 structure permits an NLS to facilitate formation of a transcriptional regulatory complex.crystallography | chromatin | nuclear localization sequence H ost cell factor-1 (HCF-1; also known as HCFC1) is a metazoan transcriptional regulator. HCF-1 was initially identified as a human coactivator for immediate-early gene expression of herpes simplex virus (HSV) by forming a complex (VP16-induced complex, VIC) with the HSV protein VP16 and the cellular transcriptional regulator Oct-1 (1). HCF-1 regulates cell-cycle progression by mediating associations between DNA-binding transcription factors and chromatin modifying complexes (2-12).Many HCF-1 proteins, including all known vertebrate HCF-1s, undergo a process of proteolytic maturation. Thus, human HCF-1 is synthesized as a 2035-aa precursor, which is cleaved and modified by O-GlcNAC transferase (13) to generate HCF-1 N and HCF-1 C subunits possessing different roles in, for example, cell-cycle regulation (14). After cleavage, the two resulting HCF-1 N and HCF-1 C subunits remain noncovalently associated via two "selfassociation sequences" called SAS1 and SAS2: SAS1 is the primary association element and its sequence is conserved in HCF-1, whereas SAS2 is secondary, less well conserved, and not considered in this study (15).SAS1 is composed of a short 43-aa HCF-1 N segment called SAS1N and a 197-aa HCF-1 C segment called SAS1C (Fig. 1A). Wilson et al. (15) suggested that SAS1C has two tandem fibronectin type 3 (Fn3) repeat structures forming a binding site for the 43-aa SAS1N to promote HCF-1 N -HCF-1 C association.To understand the mechanism of HCF-1 self-association, we have determined the crystal structure of SAS1 with the neighboring nuclear localization signal (called here SAS1-NLS). Contrary to expectation, SAS1N and SAS1C together-not SAS1C aloneform an integrated tandem Fn3 structure. Furthermore, we show that the self-association tethers the basic NLS to the HCF-1 N Kelch domain to facilitate VP16-induced complex formation. ResultsCrystal Structure of the HCF-1 SAS1 Self-Association Domain. The SAS1-NLS crystal structure (Fig. 1B) was determined to 2.7 Å resolution using the Se single-wavelength anomalous dispersion (SAD) method (Table S1 and Fig. S1). Fig. S2 details schematically...

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“…This atomic model of the P pilus explains the mechanism of rod uncoiling at the molecular level by revealing the details of an extensive inter-subunit interaction network 50 . As previously shown, the DSE interactions connecting adjacent subunits are strong hydrophobic interactions, which are topologically essential as they provide the fold-complementing interactions for each subunit in the polymer 20,21,51 . By contrast, the remainder of the subunit interaction network responsible for maintaining the helically wound quaternary structure of the pilus is not only composed of predominantly weak hydrophilic contacts but is also topologically non-essential as it is not involved in subunit folding and stability 50 .…”

Section: Structure Of the Pilus Rodmentioning

confidence: 84%

A comprehensive guide to pilus biogenesis in Gram-negative bacteria

2017

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Pili are critical virulence factors of many Gram-negative pathogens. These surface structures provide bacteria with a link to their outside environments, allowing bacteria to interact with their hosts, other surfaces or with each other. They can even provide a conduit for the exchange of information. The surface chemistries and other biophysical properties of pili are uniquely adapted to the environmental challenges faced by bacteria. The assembly of these surface structures presents a molecular engineering challenge, which bacteria have solved in a variety of unique ways. In this review, we examine recent advances in our structural understanding of various Gram-negative pilus systems and discuss their functional implications.

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Infinite Kinetic Stability against Dissociation of Supramolecular Protein Complexes through Donor Strand Complementation

Cited by 72 publications

References 42 publications

Both ATPase Domains of ClpA Are Critical for Processing of Stable Protein Structures

Both ATPase Domains of ClpA Are Critical for Processing of Stable Protein Structures

HCF-1 self-association via an interdigitated Fn3 structure facilitates transcriptional regulatory complex formation

A comprehensive guide to pilus biogenesis in Gram-negative bacteria

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