Structural and dynamic properties that govern the stability of an engineered fibronectin type III domain
Protein Engineering, Design & Selection
Structural and dynamic properties that govern the stability of an engineered fibronectin type III domain
Benjamin T. Porebski 2
Adrian A. Nickson 1
David E. Hoke 2
Morag R. Hunter 0
Liguang Zhu 3
Sheena McGowan 2
Geoffrey I. Webb 3
Ashley M. Buckle 2
0 Centre for Brain Research and Department of Pharmacology and Clinical Pharmacology, Faculty of Medical and Health Sciences, University of Auckland , Auckland , New Zealand
1 Department of Chemistry, University of Cambridge , Lensfield Road, Cambridge CB2 1EW , UK
2 Department of Biochemistry and Molecular Biology, Faculty of Medicine, School of Biomedical Sciences, Monash University , Clayton, VIC 3800 , Australia
3 Faculty of Information Technology, Monash University , Clayton, VIC 3800 , Australia
Consensus protein design is a rapid and reliable technique for the improvement of protein stability, which relies on the use of homologous protein sequences. To enhance the stability of a fibronectin type III (FN3) domain, consensus design was employed using an alignment of 2123 sequences. The resulting FN3 domain, FN3con, has unprecedented stability, with a melting temperature >100C, a GDN of 15.5 kcal mol1 and a greatly reduced unfolding rate compared with wild-type. To determine the underlying molecular basis for stability, an X-ray crystal structure of FN3con was determined to 2.0 and compared with other FN3 domains of varying stabilities. The structure of FN3con reveals significantly increased salt bridge interactions that are cooperatively networked, and a highly optimized hydrophobic core. Molecular dynamics simulations of FN3con and comparison structures show the cooperative power of electrostatic and hydrophobic networks in improving FN3con stability. Taken together, our data reveal that FN3con stability does not result from a single mechanism, but rather the combination of several features and the removal of non-conserved, unfavorable interactions. The large number of sequences employed in this study has most likely enhanced the robustness of the consensus design, which is now possible due to the increased sequence availability in the post-genomic era. These studies increase our knowledge of the molecular mechanisms that govern stability and demonstrate the rising potential for enhancing stability via the consensus method.
consensus design; fibronectin type III; FN3; molecular dynamics; stability
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There are currently several approaches employed to enhance protein
stability. The rational approach to stabilization is challenging since
it is difficult to predict the energetic and structural response to
mutation in proteins, due to inaccuracies in predictive energy
functions and the current inability to model the unfolded state (Magliery
et al., 2011). Much effort has been focused on stabilizing the native,
folded state (positive design (Dantas et al., 2003; Kuhlman, 2003;
Shah et al., 2007)) and also destabilizing the non-native states
(negative design (Richardson and Richardson, 2002; Jin et al., 2003)) via
rational design and structural comparison of thermophilic proteins
with their mesophilic counterparts (Russell et al., 1994; Russell and
Taylor, 1995; Auerbach et al., 1997; Davlieva and Shamoo, 2010;
Nakamura et al., 2010; Guelorget et al., 2011; Sundaresan et al.,
2012). Although much insight has been gained from these studies,
both approaches require structures of the target protein and or any
thermophilic orthologs, which then needs to be followed up with
extensive structural and functional analysis. These challenges are
further complicated by the context dependence of stabilizing mutations
and tend to be applicable to only a small subset of scaffolds.
An alternative approach is to utilize statistical analysis of the entire
protein fold, motif or domain of interest. This is an attractive idea
based on the hypothesis that at a given position in a multiple sequence
alignment (MSA) of homologous proteins, the respective consensus
amino acid contributes more than average to the stability of the
protein than non-consensus amino acids (Steipe et al., 1994; Lehmann
and Wyss, 2001; Magliery et al., 2011). However, the technique is
not always simple to implement. In particular, generation of MSAs
is challenging, especially in poorly conserved regions, which leads to
a large amount of noise. As most sites across a protein family are not
conserved, the most common amino acid tends to be no better than
picking a residue at random (Dantas et al., 2003; Kuhlman, 2003;
Shah et al., 2007; Magliery et al., 2011). Regardless, the efficacy of
consensus design in improving protein stability has been demonstrated
numerous times; with examples including antibodies (Steipe et al.,
1994; Richardson and Richardson, 2002; Jin et al., 2003), the
GroEL minichaperone (Wang et al., 1999), the Abp1p SH3 domain
(Maxwell and Davidson, 1998), the p53 DNA-binding domain
(Nikolova et al., 1998), fluorescent proteins (Dai et al., 2007), a (...truncated)