Molecular recognition and maturation of SOD1 by its evolutionarily destabilised cognate chaperone hCCS.

Superoxide dismutase-1 (SOD1) maturation comprises a string of posttranslational modifications which transform the nascent peptide into a stable and active enzyme. The successive folding, metal ion binding, and disulphide acquisition steps in this pathway can be catalysed through a direct interaction with the copper chaperone for SOD1 (CCS). This process confers enzymatic activity and reduces access to noncanonical, aggregation-prone states. Here, we present the functional mechanisms of human copper chaperone for SOD1 (hCCS)-catalysed SOD1 activation based on crystal structures of reaction precursors, intermediates, and products. Molecular recognition of immature SOD1 by hCCS is driven by several interface interactions, which provide an extended surface upon which SOD1 folds. Induced-fit complexation is reliant on the structural plasticity of the immature SOD1 disulphide sub-loop, a characteristic which contributes to misfolding and aggregation in neurodegenerative disease. Complexation specifically stabilises the SOD1 disulphide sub-loop, priming it and the active site for copper transfer, while delaying disulphide formation and complex dissociation. Critically, a single destabilising amino acid substitution within the hCCS interface reduces hCCS homodimer affinity, creating a pool of hCCS available to interact with immature SOD1. hCCS substrate specificity, segregation between solvent and biological membranes, and interaction transience are direct results of this substitution. In this way, hCCS-catalysed SOD1 maturation is finessed to minimise copper wastage and reduce production of potentially toxic SOD1 species.

Heterotypic Coiled-Coil Formation is Essential for the Correct Assembly of the Septin Heterofilament.

Protein-protein interactions play a critical role in promoting the stability of protein quaternary structure and in the assembly of large macromolecular complexes. What drives the stabilization of such assemblies is a central question in biology. A limiting factor in fully understanding such systems is the transient nature of many complexes, making structural studies difficult. Septins comprise a conserved family of guanine nucleotide binding proteins that polymerize in the form of heterofilaments. In structural terms, they have a common organization: a central GTPase domain, an N-terminal domain, and a C-terminal domain; the latter is predicted to form a coiled coil. Currently, even for the best characterized human septin heterocomplex (SEPT2/SEPT6/SEPT7), the role of C-terminal domain is not fully established, and this is partly due to the absence of electron density for the C-terminal domains in the x-ray structure. Here we present results on the homo/heterotypical affinity for the C-terminal domains of human septins belonging to the SEPT6 and SEPT7 groups (SEPT6C/8C/10C/11C and SEPT7C, respectively) and provide clear evidence that this domain determines the preference for heterotypic interactions at one specific interface during the assembly of the heterofilament. This observation has wider implications where macromolecular assemblies are defined by coiled-coil protein interactions.

Automated molecule editing in molecular design.

The ability to modify chemical structures in an automated and controlled manner is useful in molecular design. This Perspective introduces the MUDO molecule editor and shows how automated molecule editing can be used to standardize structures, enumerate tautomeric and ionization states, identify matched molecular pairs. Unlike its predecessor Leatherface, MUDO can also process 3D structures and this capability can be used to link non-covalently docked ligands to proteins.

Orientational Ambiguity in Septin Coiled Coils and its Structural Basis.

Septins are an example of subtle molecular recognition whereby different paralogues must correctly assemble into functional filaments important for essential cellular events such as cytokinesis. Most possess C-terminal domains capable of forming coiled coils which are believed to be involved in filament formation and bundling. Here, we report an integrated structural approach which aims to unravel their architectural diversity and in so doing provide direct structural information for the coiled-coil regions of five human septins. Unexpectedly, we encounter dimeric structures presenting both parallel and antiparallel arrangements which are in consonance with molecular modelling suggesting that both are energetically accessible. These sequences therefore code for two metastable states of different orientations which employ different but overlapping interfaces. The antiparallel structures present a mixed coiled-coil interface, one side of which is dominated by a continuous chain of core hydrophilic residues. This unusual type of coiled coil could be used to expand the toolkit currently available to the protein engineer for the design of previously unforeseen coiled-coil based assemblies. Within a physiological context, our data provide the first atomic details related to the assumption that the parallel orientation is likely formed between septin monomers from the same filament whilst antiparallelism may participate in the widely described interfilament cross bridges necessary for higher order structures and thereby septin function.