The structural and energetic basis for high selectivity in a high-affinity protein-protein interaction

Abstract

High-affinity, high-selectivity protein-protein interactions that are critical for cell survival present an evolutionary paradox: How does selectivity evolve when acquired mutations risk a lethal loss of high-affinity binding? A detailed understanding of selectivity in such complexes requires structural information on weak, noncognate complexes which can be difficult to obtain due to their transient and dynamic nature. Using NMR-based docking as a guide, we deployed a disulfide-trapping strategy on a noncognate complex between the colicin E9 endonuclease (E9 DNase) and immunity protein 2 (Im2), which is seven orders of magnitude weaker binding than the cognate femtomolar E9 DNase-Im9 interaction. The 1.77 Å crystal structure of the E9 DNase-Im2 complex reveals an entirely noncovalent interface where the intersubunit disulfide merely supports the crystal lattice. In combination with computational alanine scanning of interfacial residues, the structure reveals that the driving force for binding is so strong that a severely unfavorable specificity contact is tolerated at the interface and as a result the complex becomes weakened through "frustration." As well as rationalizing past mutational and thermodynamic data, comparing our noncognate structure with previous cognate complexes highlights the importance of loop regions in developing selectivity and accentuates the multiple roles of buried water molecules that stabilize, ameliorate, or aggravate interfacial contacts. The study provides direct support for dual-recognition in colicin DNase-Im protein complexes and shows that weakened noncognate complexes are primed for high-affinity binding, which can be achieved by economical mutation of a limited number of residues at the interface.colicins | crystallography | disulfide-trapping | specificity | frustration S pecificity in protein-protein interactions (PPIs) is critical for the organization of macromolecular complexes involved in all aspects of cellular homeostasis and differentiation. Within the vast interaction networks in cells there is significant redundancy, with many proteins acting as "hubs" that recognize multiple binding partners (1), and yet also significant discrimination (2). The factors that tip the balance in favor of specificity or promiscuity in PPIs while not clear are coming under increasing scrutiny (3). Understanding this balance is critical both from fundamental and applied perspectives. Protein therapeutics are finding increasing use in medicine but off-target effects can have disastrous consequences (4). High-affinity, high-selectivity binding is therefore an essential goal in such engineered platforms. Advances in defining the molecular and thermodynamic basis for binding affinity have been made in a multitude of natural and engineered/designed PPIs (5-7), but our molecular knowledge of specificity remains rudimentary. In large part this is due to the lack of highresolution structural information on weak and transient proteinprotein complexes that are evolutionarily related to a cognate hig...

NMR Characterisation of the Relationship between Frustration and the Excited State of Im7

“…41 In contrast to this, the X-ray structure of the non-cognate Im2-E9 DNase complex shows that there are highly frustrated contacts that account for a sizeable proportion of the reduced affinity that the E9 DNase has for Im2 compared with Im9. 42 NMR studies of L53A/I54A Im7 ⁎ in its free state and bound to the DNase of colicin E7…”

Structural basis for specificity and promiscuity in a carrier protein/enzyme system from the sulfur cycle

“…Proteins that engage in promiscuous interactions commonly recognize a partner protein using a surface binding site that is well-separated from the site of functional interaction (sometimes termed "dual recognition") (27,28). Distributing the binding interaction either partly or wholly to a distal site reduces the number of specific interactions that the functional domain needs to make with the partner protein.…”

Structural basis for the recognition of the scaffold protein Frmpd4/Preso1 by the TPR domain of the adaptor protein LGN

“…5d) and in the Pins-dInsc complex (data not shown). It is known that water molecules can stabilize the complex between a protein and its ligand by hydrogen bonding to the two components and can also sculpt the protein surface to improve interface complementarity (Barillari et al, 2007;Meenan et al, 2010).…”