Research Article: Unusually high mechanical stability of bacterial adhesin extender domains having calcium clamps

Date Published: April 4, 2017

Publisher: Public Library of Science

Author(s): Anneloes S. Oude Vrielink, Tyler D. R. Vance, Arthur M. de Jong, Peter L. Davies, Ilja K. Voets, Miklos S. Kellermayer.


To gain insight into the relationship between protein structure and mechanical stability, single molecule force spectroscopy experiments on proteins with diverse structure and topology are needed. Here, we measured the mechanical stability of extender domains of two bacterial adhesins MpAFP and MhLap, in an atomic force microscope. We find that both proteins are remarkably stable to pulling forces between their N- and C- terminal ends. At a pulling speed of 1 μm/s, the MpAFP extender domain fails at an unfolding force Fu = 348 ± 37 pN and MhLap at Fu = 306 ± 51 pN in buffer with 10 mM Ca2+. These forces place both extender domains well above the mechanical stability of many other β-sandwich domains in mechanostable proteins. We propose that the increased stability of MpAFP and MhLap is due to a combination of both hydrogen bonding between parallel terminal strands and intra-molecular coordination of calcium ions.

Partial Text

Single molecule force spectroscopy (SMFS) has been used to measure the mechanical stability of proteins and to elucidate their underlying molecular mechanisms. [1, 2] Tens of proteins have been subjected to force spectroscopy measurements, which have revealed that β-sheet proteins tend to be more stable, relative to α-helical proteins. [3, 4] Several of the strongest natural proteins reported to date have β-sandwich folds, and are often repetitive domains of larger, extracellular proteins, where high mechanical strength is an asset for proper function in the face of environmental pressures. For example, the Ig-like domains 27 and 32 of the intracellular human muscle protein titin unfold at roughly 200 pN and 300 pN, respectively (400 nm/s pulling speed) [5, 6], while cohesin I modules from the extracellular Clostridium scaffoldins, CipA and CipC connecting region, unfold at forces above 400 pN (400 nm/s pulling speed). [7] A key determinant of the high mechanical stability of these proteins is the presence of hydrogen bonds between parallel β-strands found at the termini of each repeat, which form the mechanical clamp: a structural region in a protein that is responsible for the enhanced resistance to stretching. [8] The cohesin I modules from CipA and CipC (known as c7A and c1C, respectively) have two mechanical clamps in tandem, which would explain their substantially higher mechanical stability relative to the titin repeats which only contain a single clamp. The resistance to shearing by parallel, terminal β-strands has also been shown to enhance the mechanical stability in non-mechanical proteins, such as protein L and GB1. [9, 10] Furthermore, this motif has been used to single out proteins with high mechanical stability in the protein data bank (PDB). [11] Coarse-grained molecular dynamics simulations with protein structures deposited in the PDB, have led to the classification of several types of mechanical clamp motifs including shear (of which there are parallel-, antiparallel-, disconnected-, and supported-), delocalized, zipper and torsion clamp motifs. [12–14] Apart from proteins with cysteine slipknots, the scaffoldins c1C and c7A were among the strongest proteins, with a high predicted mechanical stability that could be experimentally verified. [7]

AFM single molecule force spectroscopy experiments were performed at 10 mM free Ca2+ concentrations to determine the mechanical strength of the extender domains (region II) of two Ca2+-dependent bacterial adhesins: MpAFP and MhLap. We find that RII of both bacterial adhesins has an exceptionally high mechanical stability at 10 mM Ca2+ with unfolding forces Fu = 348 ± 37 pN and Fu = 306 ± 51 pN respectively at a pulling speed of 1 μm/s. The stability of MpAFP RII is drastically reduced at 30 μM Ca2+ where the protein is only partially folded. While these proteins are not as strong as the cohesion I modules of CipA and CipC, they still have higher unfolding forces than many single-mechanical clamp proteins like titin I27. The impressive strength of MpAFP RII and MhLap RII is attributed to a combination of a classical mechanical clamp region and complementary calcium clamps that anchor the terminal strands via coordination to neighboring secondary structure elements. Together, these molecular attributes allow the adhesins’ host bacteria to retain advantageous positions in their environments, in the face of strong shear forces.




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