Mutant libraries reveal negative design shielding proteins from supramolecular self-assembly and relocalization in cells.

Understanding the molecular consequences of mutations in proteins is essential to map genotypes to phenotypes and interpret the increasing wealth of genomic data. While mutations are known to disrupt protein structure and function, their potential to create new structures and localization phenotypes has not yet been mapped to a sequence space. To map this relationship, we employed two homo-oligomeric protein complexes in which the internal symmetry exacerbates the impact of mutations. We mutagenized three surface residues of each complex and monitored the mutations' effect on localization and assembly phenotypes in yeast cells. While surface mutations are classically viewed as benign, our analysis of several hundred mutants revealed they often trigger three main phenotypes in these proteins: nuclear localization, the formation of puncta, and fibers. Strikingly, more than 50% of random mutants induced one of these phenotypes in both complexes. Analyzing the mutant's sequences showed that surface stickiness and net charge are two key physicochemical properties associated with these changes. In one complex, more than 60% of mutants self-assembled into fibers. Such a high frequency is explained by negative design: charged residues shield the complex from self-interacting with copies of itself, and the sole removal of the charges induces its supramolecular self-assembly. A subsequent analysis of several other complexes targeted with alanine mutations suggested that such negative design is common. These results highlight that minimal perturbations in protein surfaces' physicochemical properties can frequently drive assembly and localization changes in a cellular context.

Not Going with the Flow: How Cells Adapt Internal Physics.

Defining the principles underlying the organization of biomolecules within cells is a key challenge of current cell biology research. Persson et al. now identify a powerful layer of regulation that allows cells to decouple diffusion from temperature by modulating their intracellular viscosity. This so-called viscoadaptation is mediated through trehalose and glycogen activities, which alter diffusion dynamics and self-assembly propensity inside the cell globally.

Geometric description of self-interaction potential in symmetric protein complexes.

Proteins can self-associate with copies of themselves to form symmetric complexes called homomers. Homomers are widespread in all kingdoms of life and allow for unique geometric and functional properties, as reflected in viral capsids or allostery. Once a protein forms a homomer, however, its internal symmetry can compound the effect of point mutations and trigger uncontrolled self-assembly into high-order structures. We identified mutation hot spots for supramolecular assembly, which are predictable by geometry. Here, we present a dataset of descriptors that characterize these hot spot positions both geometrically and chemically, as well as computer scripts allowing the calculation and visualization of these properties for homomers of choice. Since the biological relevance of homomers is not readily available from their X-ray crystallographic structure, we also provide reliability estimates obtained by methods we recently developed. These data have implications in the study of disease-causing mutations, protein evolution and can be exploited in the design of biomaterials.

Infinite Assembly of Folded Proteins in Evolution, Disease, and Engineering.

Mutations and changes in a protein's environment are well known for their potential to induce misfolding and aggregation, including amyloid formation. Alternatively, such perturbations can trigger new interactions that lead to the polymerization of folded proteins. In contrast to aggregation, this process does not require misfolding and, to highlight this difference, we refer to it as agglomeration. This term encompasses the amorphous assembly of folded proteins as well as the polymerization in one, two, or three dimensions. We stress the remarkable potential of symmetric homo-oligomers to agglomerate even by single surface point mutations, and we review the double-edged nature of this potential: how aberrant assemblies resulting from agglomeration can lead to disease, but also how agglomeration can serve in cellular adaptation and be exploited for the rational design of novel biomaterials.

Proteins evolve on the edge of supramolecular self-assembly.

The self-association of proteins into symmetric complexes is ubiquitous in all kingdoms of life. Symmetric complexes possess unique geometric and functional properties, but their internal symmetry can pose a risk. In sickle-cell disease, the symmetry of haemoglobin exacerbates the effect of a mutation, triggering assembly into harmful fibrils. Here we examine the universality of this mechanism and its relation to protein structure geometry. We introduced point mutations solely designed to increase surface hydrophobicity among 12 distinct symmetric complexes from Escherichia coli. Notably, all responded by forming supramolecular assemblies in vitro, as well as in vivo upon heterologous expression in Saccharomyces cerevisiae. Remarkably, in four cases, micrometre-long fibrils formed in vivo in response to a single point mutation. Biophysical measurements and electron microscopy revealed that mutants self-assembled in their folded states and so were not amyloid-like. Structural examination of 73 mutants identified supramolecular assembly hot spots predictable by geometry. A subsequent structural analysis of 7,471 symmetric complexes showed that geometric hot spots were buffered chemically by hydrophilic residues, suggesting a mechanism preventing mis-assembly of these regions. Thus, point mutations can frequently trigger folded proteins to self-assemble into higher-order structures. This potential is counterbalanced by negative selection and can be exploited to design nanomaterials in living cells.

Symmetry breaking in homo-oligomers: the curious case of mega-hemocyanin.

Mega-hemocyanin is a 13.5 MDa oxygen transporter found in snails. It is built from three stacked rings involving ten subunits each. The cryo-EM structure of the complex presented by Gatsogiannis and colleagues in this issue of Structure revealed an unexpected breaking of 5-fold symmetry in the central ring and a nonequivalent packing of the subunits.

Probing the mutational interplay between primary and promiscuous protein functions: a computational-experimental approach.

Protein promiscuity is of considerable interest due its role in adaptive metabolic plasticity, its fundamental connection with molecular evolution and also because of its biotechnological applications. Current views on the relation between primary and promiscuous protein activities stem largely from laboratory evolution experiments aimed at increasing promiscuous activity levels. Here, on the other hand, we attempt to assess the main features of the simultaneous modulation of the primary and promiscuous functions during the course of natural evolution. The computational/experimental approach we propose for this task involves the following steps: a function-targeted, statistical coupling analysis of evolutionary data is used to determine a set of positions likely linked to the recruitment of a promiscuous activity for a new function; a combinatorial library of mutations on this set of positions is prepared and screened for both, the primary and the promiscuous activities; a partial-least-squares reconstruction of the full combinatorial space is carried out; finally, an approximation to the Pareto set of variants with optimal primary/promiscuous activities is derived. Application of the approach to the emergence of folding catalysis in thioredoxin scaffolds reveals an unanticipated scenario: diverse patterns of primary/promiscuous activity modulation are possible, including a moderate (but likely significant in a biological context) simultaneous enhancement of both activities. We show that this scenario can be most simply explained on the basis of the conformational diversity hypothesis, although alternative interpretations cannot be ruled out. Overall, the results reported may help clarify the mechanisms of the evolution of new functions. From a different viewpoint, the partial-least-squares-reconstruction/Pareto-set-prediction approach we have introduced provides the computational basis for an efficient directed-evolution protocol aimed at the simultaneous enhancement of several protein features and should therefore open new possibilities in the engineering of multi-functional enzymes.

How many ionizable groups can sit on a protein hydrophobic core?

Full or partial burial of ionizable groups in the hydrophobic interior of proteins underlies the large modulation in group properties (modified pK value, high nucleophilicity, enhanced capability of interaction with chemical moieties of the substrate, etc.) linked to biological function. Indeed, the few internal ionizable residues found in proteins are known to play important functional roles in catalysis and, in general, in energy transduction processes. However, ionizable-group burial is expected to be seriously disruptive and, it is important to note, most functional sites contain not just one, but several ionizable residues. Hence, the adaptations involved in the development of function in proteins (through in vitro engineering or during the course of natural evolution) are not fully understood. Here, we explore experimentally how proteins respond to the accumulation of hydrophobic-to-ionizable residue substitutions. For this purpose, we have constructed a combinatorial library targeting a hydrophobic cluster in a consensus-engineered, stabilized form of a small model protein. Contrary to naïve expectation, half of the variants randomly selected from the library are soluble, folded, and active, despite including up to four mutations. Furthermore, for these variants, the dependence of stability with the number of mutations is not synergistic and catastrophic, but smooth and approximately linear. Clearly, stabilized protein scaffolds may be robust enough to withstand many disruptive hydrophobic-to-ionizable residue mutations, even when they are introduced in the same region of the structure. These results should be relevant for protein engineering and may have implications for the understanding of the early evolution of enzymes.