Solving the structure of<i>Escherichia coli</i>elongation factor Tu using a twinned data set

Acinetobacter baumannii is an important nosocomial pathogen, causing a variety of opportunistic infections of the skin, soft tissues and wounds, urinary tract infections, secondary meningitis, pneumonia and bacteremia. Over 63% of A. baumannii infections occurring in the United States are caused by multidrug resistant isolates, and pan-resistant isolates have begun to emerge that are resistant to all clinically relevant antibiotics. The complement system represents the first line of defense against invading pathogens. However, many A. baumannii isolates, especially those causing severe bacteremia are resistant to complement-mediated killing, though the underlying mechanisms remain poorly understood. Here we show for the first time that A. baumannii binds host-derived plasminogen and we identify the translation elongation factor Tuf as a moonlighting plasminogen-binding protein that is exposed on the outer surface of A. baumannii. Binding of plasminogen to Tuf is at least partly dependent on lysine residues and ionic interactions. Plasminogen, once bound to Tuf can be converted to active plasmin and proteolytically degrade fibrinogen as well as the key complement component C3b. Thus, Tuf acts as a multifunctional protein that may contribute to virulence of A. baumannii by aiding in dissemination and evasion of the complement system.

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“…Fig 8A shows the 3D-structure of elongation factor Tuf of E . coli [ 45 ]. Conserved lysine residues are highlighted in blue.…”

Section: Resultsmentioning

confidence: 99%

“…(B) Predicted charge distribution across the Tuf protein. Fig was created using PyMOL, Version 1.3 and is based on PDB file 2FX3 [ 45 ].…”

Section: Resultsmentioning

confidence: 99%

Translation Elongation Factor Tuf of Acinetobacter baumannii Is a Plasminogen-Binding Protein

Zipfel

Kraiczy

2015

PLoS ONE

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“…A search for 'twinned' in a subset of the PDB in which sequence homologues with >90% identical residues were removed identified 184 entries. Inspection reveals that many of these are close to perfectly twinned, as is the case for the first four entries in the list: Escherichia coli elongation factor Tu (Heffron et al, 2006), phospholipase A 2 from the venom of Ophiophagus hannah (Xu et al, 2003), a domain-opened mutant (R121D) of the human lactoferrin N-lobe (Jameson et al, 2002) and calciumdepleted human C-reactive protein (Ramadan et al, 2002).…”

Section: Discussionmentioning

confidence: 90%

“…For the PSI data, an additional problem is that the data are low resolution (8.7 Å ), which prevents structure solution. In principle, structure solution from twinned data would allow a reference data set based on model intensities to be obtained; in practice, this is very difficult and is unlikely to succeed for data obtained by serial crystallography: working with (almost) perfectly twinned data is already difficult in conventional crystallography (see, for example, Jameson et al, 2002;Ramadan et al, 2002;Heffron et al, 2006;Xu et al, 2003).…”

Section: Introductionmentioning

confidence: 99%

Breaking the indexing ambiguity in serial crystallography

Brehm

Diederichs

2013

Acta Crystallogr D Biol Crystallogr

101

110

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In serial crystallography, a very incomplete partial data set is obtained from each diffraction experiment (a `snapshot'). In some space groups, an indexing ambiguity exists which requires that the indexing mode of each snapshot needs to be established with respect to a reference data set. In the absence of such re‐indexing information, crystallographers have thus far resorted to a straight merging of all snapshots, yielding a perfectly twinned data set of higher symmetry which is poorly suited for structure solution and refinement. Here, two algorithms have been designed for assembling complete data sets by clustering those snapshots that are indexed in the same way, and they have been tested using 15 445 snapshots from photosystem I [Chapman et al. (2011), Nature (London), 470, 73–77] and with noisy model data. The results of the clustering are unambiguous and enabled the construction of complete data sets in the correct space group P63 instead of (twinned) P6322 that researchers have been forced to use previously in such cases of indexing ambiguity. The algorithms thus extend the applicability and reach of serial crystallography.

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“…Recognition of EF‐Tu domains by human IgG. (A) Three‐dimensional structure of EF‐Tu, based on PDB 1dg1 , showing the organization of EF‐Tu domains 1‐3. (B) Recombinant NTHi EF‐Tu was analyzed by SDS‐PAGE to determine the fragmentation pattern from CNBr cleavage assay.…”

Section: Resultsmentioning

confidence: 99%

Anti‐EF‐Tu IgG titers increase with age and may contribute to protection against the respiratory pathogen Haemophilus influenzae

Thofte

Kaur

et al. 2019

Eur J Immunol

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Non‐typeable Haemophilus influenzae (NTHi) is a pathogen that commonly colonizes the nasopharynx of preschool children, causing opportunistic infections including acute otitis media (AOM). Patients suffering from chronic obstructive pulmonary disease (COPD) are persistently colonized with NTHi and occasionally suffer from exacerbations by the bacterium leading to increased morbidity. Elongation‐factor thermo unstable (EF‐Tu), a protein critical for bacterial protein synthesis, has been found to moonlight on the surface of several bacteria. Here, we show that antibodies against NTHi EF‐Tu were present in children already at 18 months of age, and that the IgG antibody titers increased with age. Children harboring NTHi in the nasopharynx also displayed significantly higher IgG concentrations. Interestingly, children suffering from AOM had significantly higher anti‐EF‐Tu IgG levels when NTHi was the causative agent. Human sera recognized mainly the central and C‐terminal part of the EF‐Tu molecule and peptide‐based epitope mapping confirmed similar binding patterns for sera from humans and immunized mice. Immunization of BALB/c and otitis‐prone Junbo (C3H/HeH) mice promoted lower infection rates in the nasopharynx and middle ear, respectively. In conclusion, our results suggest that IgG directed against NTHi EF‐Tu may play an important role in the host immune response against NTHi.

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Solving the structure ofEscherichia colielongation factor Tu using a twinned data set

Cited by 9 publications

References 30 publications

Translation Elongation Factor Tuf of Acinetobacter baumannii Is a Plasminogen-Binding Protein

Translation Elongation Factor Tuf of Acinetobacter baumannii Is a Plasminogen-Binding Protein

Breaking the indexing ambiguity in serial crystallography

Anti‐EF‐Tu IgG titers increase with age and may contribute to protection against the respiratory pathogen Haemophilus influenzae

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