Computational design of transmembrane pores.

Transmembrane channels and pores have key roles in fundamental biological processes1 and in biotechnological applications such as DNA nanopore sequencing2-4, resulting in considerable interest in the design of pore-containing proteins. Synthetic amphiphilic peptides have been found to form ion channels5,6, and there have been recent advances in de novo membrane protein design7,8 and in redesigning naturally occurring channel-containing proteins9,10. However, the de novo design of stable, well-defined transmembrane protein pores that are capable of conducting ions selectively or are large enough to enable the passage of small-molecule fluorophores remains an outstanding challenge11,12. Here we report the computational design of protein pores formed by two concentric rings of α-helices that are stable and monodisperse in both their water-soluble and their transmembrane forms. Crystal structures of the water-soluble forms of a 12-helical pore and a 16-helical pore closely match the computational design models. Patch-clamp electrophysiology experiments show that, when expressed in insect cells, the transmembrane form of the 12-helix pore enables the passage of ions across the membrane with high selectivity for potassium over sodium; ion passage is blocked by specific chemical modification at the pore entrance. When incorporated into liposomes using in vitro protein synthesis, the transmembrane form of the 16-helix pore-but not the 12-helix pore-enables the passage of biotinylated Alexa Fluor 488. A cryo-electron microscopy structure of the 16-helix transmembrane pore closely matches the design model. The ability to produce structurally and functionally well-defined transmembrane pores opens the door to the creation of designer channels and pores for a wide variety of applications.

High thermodynamic stability of parametrically designed helical bundles.

We describe a procedure for designing proteins with backbones produced by varying the parameters in the Crick coiled coil-generating equations. Combinatorial design calculations identify low-energy sequences for alternative helix supercoil arrangements, and the helices in the lowest-energy arrangements are connected by loop building. We design an antiparallel monomeric untwisted three-helix bundle with 80-residue helices, an antiparallel monomeric right-handed four-helix bundle, and a pentameric parallel left-handed five-helix bundle. The designed proteins are extremely stable (extrapolated ΔGfold > 60 kilocalories per mole), and their crystal structures are close to those of the design models with nearly identical core packing between the helices. The approach enables the custom design of hyperstable proteins with fine-tuned geometries for a wide range of applications.

Insight into the architecture of the NuRD complex: structure of the RbAp48-MTA1 subcomplex.

The nucleosome remodeling and deacetylase (NuRD) complex is a widely conserved transcriptional co-regulator that harbors both nucleosome remodeling and histone deacetylase activities. It plays a critical role in the early stages of ES cell differentiation and the reprogramming of somatic to induced pluripotent stem cells. Abnormalities in several NuRD proteins are associated with cancer and aging. We have investigated the architecture of NuRD by determining the structure of a subcomplex comprising RbAp48 and MTA1. Surprisingly, RbAp48 recognizes MTA1 using the same site that it uses to bind histone H4, showing that assembly into NuRD modulates RbAp46/48 interactions with histones. Taken together with other results, our data show that the MTA proteins act as scaffolds for NuRD complex assembly. We further show that the RbAp48-MTA1 interaction is essential for the in vivo integration of RbAp46/48 into the NuRD complex.

Selectivity and self-assembly in the control of a bacterial toxin by an antitoxic noncoding RNA pseudoknot.

Bacterial small RNAs perform numerous regulatory roles, including acting as antitoxic components in toxin-antitoxin systems. In type III toxin-antitoxin systems, small processed RNAs directly antagonize their toxin protein partners, and in the systems characterized the toxin and antitoxin components together form a trimeric assembly. In the present study, we sought to define how the RNA antitoxin, ToxI, inhibits its potentially lethal protein partner, ToxN. We show through cross-inhibition experiments with the ToxIN systems from Pectobacterium atrosepticum (ToxIN(Pa)) and Bacillus thuringiensis (ToxIN(Bt)) that ToxI RNAs are highly selective enzyme inhibitors. Both systems have an "addictive" plasmid maintenance phenotype. We demonstrate that ToxI(Pa) can inhibit ToxN(Pa) in vitro both in its processed form and as a repetitive precursor RNA, and this inhibition is linked to the self-assembly of the trimeric complex. Inhibition and self-assembly are both mediated entirely by the ToxI(Pa) RNA, with no requirement for cellular factors or exogenous energy. Finally, we explain the origins of ToxI antitoxin selectivity through our crystal structure of the ToxIN(Bt) complex. Our results show how a processed RNA pseudoknot can inhibit a deleterious protein with exquisite molecular specificity and how these self-contained and addictive RNA-protein pairs can confer different adaptive benefits in their bacterial hosts.

Identification and classification of bacterial Type III toxin-antitoxin systems encoded in chromosomal and plasmid genomes.

Toxin-antitoxin systems are widespread in bacteria and archaea. They perform diverse functional roles, including the generation of persistence, maintenance of genetic loci and resistance to bacteriophages through abortive infection. Toxin-antitoxin systems have been divided into three types, depending on the nature of the interacting macromolecules. The recently discovered Type III toxin-antitoxin systems encode protein toxins that are inhibited by pseudoknots of antitoxic RNA, encoded by short tandem repeats upstream of the toxin gene. Recent studies have identified the range of Type I and Type II systems within current sequence databases. Here, structure-based homology searches were combined with iterative protein sequence comparisons to obtain a current picture of the prevalence of Type III systems. Three independent Type III families were identified, according to toxin sequence similarity. The three families were found to be far more abundant and widespread than previously known, with examples throughout the Firmicutes, Fusobacteria and Proteobacteria. Functional assays confirmed that representatives from all three families act as toxin-antitoxin loci within Escherichia coli and at least two of the families confer resistance to bacteriophages. This study shows that active Type III toxin-antitoxin systems are far more diverse than previously known, and suggests that more remain to be identified.

A processed noncoding RNA regulates an altruistic bacterial antiviral system.

The ≥ 10³° bacteriophages on Earth relentlessly drive adaptive coevolution, forcing the generation of protective mechanisms in their bacterial hosts. One such bacterial phage-resistance system, ToxIN, consists of a protein toxin (ToxN) that is inhibited in vivo by a specific RNA antitoxin (ToxI); however, the mechanisms for this toxicity and inhibition have not been defined. Here we present the crystal structure of the ToxN-ToxI complex from Pectobacterium atrosepticum, determined to 2.75-Å resolution. ToxI is a 36-nucleotide noncoding RNA pseudoknot, and three ToxI monomers bind to three ToxN monomers to generate a trimeric ToxN-ToxI complex. Assembly of this complex is mediated entirely through extensive RNA-protein interactions. Furthermore, a 2'-3' cyclic phosphate at the 3' end of ToxI, and catalytic residues, identify ToxN as an endoRNase that processes ToxI from a repetitive precursor but is regulated by its own catalytic product.

The molecular origins of specificity in the assembly of a multienzyme complex.

The pyruvate dehydrogenase (PDH) multienzyme complex is central to oxidative metabolism. We present the first crystal structure of a complex between pyruvate decarboxylase (E1) and the peripheral subunit binding domain (PSBD) of the dihydrolipoyl acetyltransferase (E2). The interface is dominated by a "charge zipper" of networked salt bridges. Remarkably, the PSBD uses essentially the same zipper to alternately recognize the dihydrolipoyl dehydrogenase (E3) component of the PDH assembly. The PSBD achieves this dual recognition largely through the addition of a network of interfacial water molecules unique to the E1-PSBD complex. These structural comparisons illuminate our observations that the formation of this water-rich E1-E2 interface is largely enthalpy driven, whereas that of the E3-PSBD complex (from which water is excluded) is entropy driven. Interfacial water molecules thus diversify surface complementarity and contribute to avidity, enthalpically. Additionally, the E1-PSBD structure provides insight into the organization and active site coupling within the approximately 9 MDa PDH complex.