Summary Here we report a novel mechanism of PDZ (PSD-95/Dlg/ZO-1) domain regulation that distorts a conserved element of PDZ ligand recognition. The polarity regulator Par-6 assembles a conserved multi-protein complex and is directly modulated by the Rho GTPase Cdc42. Cdc42 binds the adjacent Cdc42/Rac interactive binding (CRIB) and PDZ domains of Par-6, increasing C-terminal ligand binding affinity 10-fold. By solving structures of the isolated PDZ domain and a disulfide-stabilized CRIB-PDZ, we detected a conformational switch that controls affinity by altering the configuration of the conserved ‘GLGF’ loop. As a result, lysine 165 is displaced from the PDZ core by an adjacent hydrophobic residue, disrupting coordination of the PDZ ligand binding cleft. Stabilization of the CRIB:PDZ interface restores K165 to its canonical location in the binding pocket. We conclude that a unique ‘dipeptide switch’ in the Par-6 PDZ transmits a signal for allosteric activation to the ligand binding pocket.

To form and maintain organized tissues, multicellular organisms orient their mitotic spindles relative to neighboring cells. A molecular complex scaffolded by the GK protein-interaction domain (GKPID) mediates spindle orientation in diverse animal taxa by linking microtubule motor proteins to a marker protein on the cell cortex localized by external cues. Here we illuminate how this complex evolved and commandeered control of spindle orientation from a more ancient mechanism. The complex was assembled through a series of molecular exploitation events, one of which – the evolution of GKPID’s capacity to bind the cortical marker protein – can be recapitulated by reintroducing a single historical substitution into the reconstructed ancestral GKPID. This change revealed and repurposed an ancient molecular surface that previously had a radically different function. We show how the physical simplicity of this binding interface enabled the evolution of a new protein function now essential to the biological complexity of many animals.DOI: http://dx.doi.org/10.7554/eLife.10147.001

New protein functions can require complex sequence changes, but the minimal path is not well understood. The guanylate kinase enzyme (GK enz ), which catalyzes phosphotransfer from ATP to GMP, evolved into the GK domain (GK dom ), a protein-binding domain found in membrane associate guanylate kinases that function in mitotic spindle orientation and cell adhesion. Using an induced polarity assay for GK dom function, we show that a single serine to proline mutation is sufficient to switch extant GK enz into a functional GK dom . The mutation blocks catalysis (GK enz function) but allows protein binding and spindle orientation (GK dom function). Furthermore, whereas the GK enz undergoes a large closing motion upon GMP binding, fluorescence quenching and NMR demonstrate that the S → P mutation inhibits GMP-induced GK movements. Disrupting GK closing with a mutation at a different position also leads to GK dom function, suggesting that blocking the GK enz closing motion is sufficient for functional conversion of GK enz to GK dom . Although subtle changes in protein function can require complex sequence paths, our work shows that entirely new functions can arise from single mutations that alter protein dynamics.protein interactions | neofunctionalization | protein engineering | molecular evolution P roteins perform remarkably diverse functions including catalysis, small molecule binding, and protein recognition. Molecular evolution seeks to understand how the diversity of known protein functions arose, whereas the goal of protein engineering is to create new ones (1). An evolutionary process for creating new protein functions is the divergent evolution of duplicated genes, in which gene duplication is followed by functional evolution [neofunctionalization; (2)]. However, loss of function (pseudogene formation or nonfunctionalization) competes with functional evolution and may be much more likely (3). The degree to which neofunctionalization can "win out" over pseudogene formation depends on the number of mutations required to attain the new function and the possible paths in sequence space to the new function. Thus, neofunctionalization is a competitive process that is dependent on the number of required mutations and how protein function is achieved. Insight into the mechanisms of neofunctionalization should improve our ability to develop previously undescribed protein functions.We examined neofunctionalization in the context of a dramatic functional change: creation of a mitotic spindle-orienting protein from a nucleotide kinase within the membrane associated guanylate kinase (MAGUK) family of proteins. MAGUKs are proteins that mediate intricate multicellular functions such as cell junction formation and mitotic spindle orientation and have been found only in metazoans and choanoflagellates (4, 5).

Par-6 is a scaffold protein that organizes other proteins into a complex required to initiate and maintain cell polarity. Cdc42-GTP binds the CRIB module of Par-6 and alters the binding affinity of the adjoining PDZ domain. Allosteric regulation of the Par-6 PDZ domain was first demonstrated using a peptide identified in a screen of typical carboxyl terminal ligands. Crumbs, a membrane protein that localizes a conserved polarity complex, was subsequently identified as a functional partner for Par-6 that likely interacts with the PDZ domain. Here we show by NMR that Par-6 binds a Crumbs carboxyl terminal peptide and report the crystal structure of the PDZ-peptide complex. The Crumbs peptide binds Par-6 more tightly than the previously studied carboxyl peptide ligand and interacts with the CRIB-PDZ module in a Cdc42-dependent manner. The Crumbs:Par-6 crystal structure reveals specific PDZ-peptide contacts that contribute to its higher affinity and Cdc42-enhanced binding. Comparisons with existing structures suggest that multiple C-terminal Par-6 ligands respond to a common conformational switch that transmits the allosteric effects of GTPase binding.

Proteins exist in a delicate balance between the native and unfolded states, where thermodynamic stability may be sacrificed to attain the flexibility required for efficient catalysis, binding or allosteric control. Par-6 regulates the Par polarity complex by transmitting a GTPase signal through the CRIB-PDZ module that alters PDZ lig-and binding. Allosteric activation of the PDZ is achieved by local rearrangement of the L164 and K165 side chains to stabilize the interdomain CRIB-PDZ interface and reposition a conserved element of the ligand binding pocket. However, microsecond to millisecond dynamics measurements revealed that L164/K165 exchange requires a larger rearrangement than expected. The margin of thermodynamic stability for the PDZ domain is modest (~3 kcal/mol) and further reduced by transient interactions with the disordered CRIB domain. Measurements of local structural stability revealed that tertiary contacts within the PDZ are disrupted by a partial unfolding transition that enables interconversion of the L/K switch. The unexpected participation of partial PDZ unfolding in the allosteric mechanism of Par-6 suggests that native-state unfolding may be essential for the function of other marginally stable proteins.