Simple yet functional phosphate-loop proteins

Abstract

Abundant and essential motifs, such as phosphate-binding loops (P-loops), are presumed to be the seeds of modern enzymes. The Walker-A P-loop is absolutely essential in modern NTPase enzymes, in mediating binding, and transfer of the terminal phosphate groups of NTPs. However, NTPase function depends on many additional active-site residues placed throughout the protein’s scaffold. Can motifs such as P-loops confer function in a simpler context? We applied a phylogenetic analysis that yielded a sequence logo of the putative ancestral Walker-A P-loop element: a β-strand connected to an α-helix via the P-loop. Computational design incorporated this element into de novo designed β-α repeat proteins with relatively few sequence modifications. We obtained soluble, stable proteins that unlike modern P-loop NTPases bound ATP in a magnesium-independent manner. Foremost, these simple P-loop proteins avidly bound polynucleotides, RNA, and single-strand DNA, and mutations in the P-loop’s key residues abolished binding. Binding appears to be facilitated by the structural plasticity of these proteins, including quaternary structure polymorphism that promotes a combined action of multiple P-loops. Accordingly, oligomerization enabled a 55-aa protein carrying a single P-loop to confer avid polynucleotide binding. Overall, our results show that the P-loop Walker-A motif can be implemented in small and simple β-α repeat proteins, primarily as a polynucleotide binding motif.

A Personal Reflection on the Chemistry‐Biology Interface

“…[9,10] Along the years, we have indeed established that conformational diversity, or structural metamorphism, is a universal principle seen throughout evolution, from the rapid and very recent evolution of enzymes that degrade pesticides introduced several decades ago, [11] to the prototypes of the earliest enzymes that emerged over 3.5 billion years ago. [12]…”

An Enzymatic Active Site Embedded in a DNA Nanostructure

“…The maturation of these technologies, together with the available software packages reached the state that allows development of nanostructures with novel functions and properties [16,17,18]. In the same time, the field of artificial enzymes attracted attention with several prominent publications on the de novo design of the catalytic systems [20][21][22][23][24][25]. Although, experimental approach, size and complexity between described systems significantly varied, they have mutual design principles, as the artificial catalytic properties are based on the introduction of modifications, on predefined positions, in a stable framework structure.…”

“…To maintain the stability of the active site, the structures of artificial enzymes are often based on extremely conserved proteins with stable secondary or tertiary structures [20,25]. When directly connected to this type of framework, the stability of the artificial catalytic site is increased, however, the design of the active site becomes limited.…”

“…When directly connected to this type of framework, the stability of the artificial catalytic site is increased, however, the design of the active site becomes limited. The framework structure, of a topdown design approach, allows for only a small number of designated places for the positioning of the key functional groups [20,23,25]. Further, introduction of the mutations in the peptide sequence can affect protein folding, and destabilise or structurally change the whole construct, and not only the functional groups involved in the artificial catalytic process.…”

Family permutation profiling identifies a dynamic protein domain as functionally tolerant to increased conformational entropy

“…This analysis reveals that new termini are differentially tolerated at diverse locations within the different AK orthologs. Circularly permuted AK were uniformly inactive when new termini were generated within the glycine rich p-loop (residues 7-15), the region of the core domain that constitutes the active site in AKs and other related kinases (42,46). In contrast, the lid (residues 128-159) and core (residues 1-30, 60-127, and 160-217) domains both varied in their tolerance to new termini across the different AK orthologs, with some positions being uniformly intolerant and other positions exhibiting tolerance that varied across the orthologs.…”