Stephen H. McLaughlin

Synthetic genetic polymers capable of heredity and evolution.

Unnatural Bases The genetic basis of all life on the planet is comprised of deoxyribonucleic acid (DNA) with four nitrogenous nucleotide bases, abbreviated to A, G, C, and T. But there are variations on this theme, and Pinheiro et al. (p. 341; see the Perspective by Joyce) describe the directed evolution of unnatural nucleic acid–like genetic polymers. Variant enzymes were developed that efficiently transcribed DNA to anhydrohexitol (HNA), cyclohexenyl (CeNA), locked (LNA), and threofuranosyl (TNA) nuceic acid analogs. Further variant enzymes were developed to reverse-transcribe these analogs back to DNA. Thus, man-made nucleic acid analogs can be designed and selected that have the potential to operate in a way analogous to the natural process of heredity and evolution. Artificial polymers of nucleic acid–like subunits not found in nature can mimic the functions of DNA and RNA. Genetic information storage and processing rely on just two polymers, DNA and RNA, yet whether their role reflects evolutionary history or fundamental functional constraints is currently unknown. With the use of polymerase evolution and design, we show that genetic information can be stored in and recovered from six alternative genetic polymers based on simple nucleic acid architectures not found in nature [xeno-nucleic acids (XNAs)]. We also select XNA aptamers, which bind their targets with high affinity and specificity, demonstrating that beyond heredity, specific XNAs have the capacity for Darwinian evolution and folding into defined structures. Thus, heredity and evolution, two hallmarks of life, are not limited to DNA and RNA but are likely to be emergent properties of polymers capable of information storage.

Structural Basis for the Recognition of Histone H4 by the Histone-Chaperone RbAp46.

Summary RbAp46 and RbAp48 (pRB-associated proteins p46 and p48, also known as RBBP7 and RBBP4, respectively) are highly homologous histone chaperones that play key roles in establishing and maintaining chromatin structure. We report here the crystal structure of human RbAp46 bound to histone H4. RbAp46 folds into a seven-bladed β propeller structure and binds histone H4 in a groove formed between an N-terminal α helix and an extended loop inserted into blade six. Surprisingly, histone H4 adopts a different conformation when interacting with RbAp46 than it does in either the nucleosome or in the complex with ASF1, another histone chaperone. Our structural and biochemical results suggest that when a histone H3/H4 dimer (or tetramer) binds to RbAp46 or RbAp48, helix 1 of histone H4 unfolds to interact with the histone chaperone. We discuss the implications of our findings for the assembly and function of RbAp46 and RbAp48 complexes.

The chromosome passenger complex is required for fidelity of chromosome transmission and cytokinesis in meiosis of mouse oocytes

The existence of two forms of the chromosome passenger complex (CPC) in the mammalian oocyte has meant that its role in female meiosis has remained unclear. Here we use loss- and gain-of function approaches to assess the meiotic functions of one of the shared components of these complexes, INCENP, and of the variable kinase subunits, Aurora B or Aurora C. We show that either the depletion of INCENP or the combined inhibition of Aurora kinases B and C activates the anaphase-promoting complex or cyclosome (APC/C) before chromosomes have properly congressed in meiosis I and also prevents cytokinesis and hence extrusion of the first polar body. Overexpression of Aurora C also advances APC/C activation and results in cytokinesis failure in a high proportion of oocytes, indicative of a dominant effect on CPC function. Together, this points to roles for the meiotic CPC in functions similar to the mitotic roles of the complex: correcting chromosome attachment to microtubules, facilitating the spindle-assembly checkpoint (SAC) function and enabling cytokinesis. Surprisingly, overexpression of Aurora B leads to a failure of APC/C activation, stabilization of securin and consequently a failure of chiasmate chromosomes to resolve – a dominant phenotype that is completely suppressed by depletion of INCENP. Taken together with the differential distribution of Aurora proteins B and C on chiasmate chromosomes, this points to differential functions of the two forms of CPC in regulating the separation of homologous chromosomes in meiosis I.

Structures of PI4KIIIβ complexes show simultaneous recruitment of Rab11 and its effectors

How to recruit membrane trafficking machinery PI4KIIIβ is a lipid kinase that underlies Golgi function and is enlisted in biological responses that require rapid delivery of membrane vesicles, such as during the extensive membrane remodeling that occurs at the end of cell division. Burke et al. determined the structure of PI4KIIIβ in a complex with the membrane trafficking GTPase Rab11a. The way in which the proteins interact gives PI4KIIIβ the ability to simultaneously recruit Rab11a and its effectors on specific membranes. Science, this issue p. 1035 A lipid kinase interacts with target membranes, a membrane trafficking guanosine triphosphatase, and its effectors simultaneously. Phosphatidylinositol 4-kinases (PI4Ks) and small guanosine triphosphatases (GTPases) are essential for processes that require expansion and remodeling of phosphatidylinositol 4-phosphate (PI4P)–containing membranes, including cytokinesis, intracellular development of malarial pathogens, and replication of a wide range of RNA viruses. However, the structural basis for coordination of PI4K, GTPases, and their effectors is unknown. Here, we describe structures of PI4Kβ (PI4KIIIβ) bound to the small GTPase Rab11a without and with the Rab11 effector protein FIP3. The Rab11-PI4KIIIβ interface is distinct compared with known structures of Rab complexes and does not involve switch regions used by GTPase effectors. Our data provide a mechanism for how PI4KIIIβ coordinates Rab11 and its effectors on PI4P-enriched membranes and also provide strategies for the design of specific inhibitors that could potentially target plasmodial PI4KIIIβ to combat malaria.

Evolution of oligomeric state through allosteric pathways that mimic ligand binding.

Introduction Evolution and design of protein complexes are frequently viewed through the lens of amino acid mutations at protein interfaces, but we showed previously that residues distant from interfaces are also commonly involved in the evolution of alternative quaternary structures. We hypothesized that in these protein families, the difference in oligomeric state is due to a change in intersubunit geometry. The indirect mutations would act by changing protein conformation and dynamics, similar to the way in which allosteric small molecules introduce functional conformational change. We refer to these substitutions as “allosteric mutations.” Allosteric mutations change oligomeric state by employing the same conformational dynamics as ligands. PyrR homologs differ by mutations, all of which are outside the tetrameric interface. A subset of these allosteric mutations can be used to engineer a shift in oligomeric state in the ancestral PyrR. Allosteric mutations act by introducing conformational change in a manner analogous to that of the allosteric ligands. Rationale In this work, we investigate the mechanism of action of allosteric mutations on oligomeric state in the PyrR family of pyrimidine operon attenuators. In this family, an entirely sequence-conserved helix that forms a tetrameric interface in the thermophilic ortholog (BcPyrR) switches to being solvent-exposed in the mesophilic ortholog (BsPyrR). This results in a homodimeric structure in which the two subunits are clearly rotated relative to their orientation in the tetramer. What is the origin of this rotation and the change in quaternary structure? To dissect the role of the 49 substitutions between BsPyrR and BcPyrR, we used ancestral sequence reconstruction in combination with structural and biophysical methods to identify a set of allosteric mutations that are responsible for this shift in conformation. We compared the conformational changes introduced by the mutations to the protein motion during allosteric regulation by guanosine monophosphate (GMP). Results We identified 11 key mutations controlling oligomeric state, all distant from the interfaces and outside ligand-binding pockets. We confirmed the role of these allosteric mutations by engineering a shift in oligomeric state in an inferred ancestral PyrR protein (intermediate in sequence between the extant orthologs). We further used the inferred ancestral states and their mutants to show that the allosteric mutations are part of a downhill adaptation of the PyrR proteins to lower temperatures. We compared the x-ray crystal structures of ancestral and engineered PyrR proteins to the free and GMP-bound structure of the mesophilic BsPyrR, which shifts its equilibrium from dimer to tetramer upon ligand binding. Binding of the allosteric molecule introduces a change in intersubunit geometry that is equivalent to the evolutionary difference in intersubunit geometry between the dimeric and tetrameric homologs. We further find that the difference in oligomeric state is coupled to the difference in intrinsic dynamics of the dimers. Finally, we used the residue-residue contact network approach to show that the residues corresponding to the allosteric mutations undergo large contact rewiring when the intersubunit geometry and, in turn, oligomeric state change, either by GMP binding or by the introduction of allosteric mutations. Conclusion We show that evolution employs the intrinsic dynamics of this protein to toggle a conformational switch in a manner similar to that of small molecules. Shifting the relative populations of different states by subtle modifications is a process central to protein function and, as shown here, also to protein evolution. This suggests that we can learn from evolution and design proteins with multiple conformational states. Evolution and design of protein complexes are almost always viewed through the lens of amino acid mutations at protein interfaces. We showed previously that residues not involved in the physical interaction between proteins make important contributions to oligomerization by acting indirectly or allosterically. In this work, we sought to investigate the mechanism by which allosteric mutations act, using the example of the PyrR family of pyrimidine operon attenuators. In this family, a perfectly sequence-conserved helix that forms a tetrameric interface is exposed as solvent-accessible surface in dimeric orthologs. This means that mutations must be acting from a distance to destabilize the interface. We identified 11 key mutations controlling oligomeric state, all distant from the interfaces and outside ligand-binding pockets. Finally, we show that the key mutations introduce conformational changes equivalent to the conformational shift between the free versus nucleotide-bound conformations of the proteins. Mutations can alter protein conformations in the same way that allosteric small molecules do. Controlling the state of dynamic proteins Small molecules that change the oligomeric state of proteins by binding to a site distant from the interface are called allosteric. They often act by taking advantage of intrinsic protein dynamics and stabilizing a particular conformation of the protein. Perica et al. show that mutations can similarly act at a distance to change protein conformation. They identified 11 mutations in an RNA- binding protein that determine whether it is stable as a dimer or a tetramer. Examination of ancestral sequences showed that the allosteric mutations are part of a downhill adaptation to lower environmental temperatures. This mechanism for modulating the oligomeric state is probably common in evolution. Science, this issue 10.1126/science.1254346

SPATA2 Links CYLD to LUBAC, Activates CYLD, and Controls LUBAC Signaling

Summary The linear ubiquitin chain assembly complex (LUBAC) regulates immune signaling, and its function is regulated by the deubiquitinases OTULIN and CYLD, which associate with the catalytic subunit HOIP. However, the mechanism through which CYLD interacts with HOIP is unclear. We here show that CYLD interacts with HOIP via spermatogenesis-associated protein 2 (SPATA2). SPATA2 interacts with CYLD through its non-canonical PUB domain, which binds the catalytic CYLD USP domain in a CYLD B-box-dependent manner. Significantly, SPATA2 binding activates CYLD-mediated hydrolysis of ubiquitin chains. SPATA2 also harbors a conserved PUB-interacting motif that selectively docks into the HOIP PUB domain. In cells, SPATA2 is recruited to the TNF receptor 1 signaling complex and is required for CYLD recruitment. Loss of SPATA2 increases ubiquitination of LUBAC substrates and results in enhanced NOD2 signaling. Our data reveal SPATA2 as a high-affinity binding partner of CYLD and HOIP, and a regulatory component of LUBAC-mediated NF-κB signaling.

Structure of the SAS-6 cartwheel hub from Leishmania major

Centrioles are cylindrical cell organelles with a ninefold symmetric peripheral microtubule array that is essential to template cilia and flagella. They are built around a central cartwheel assembly that is organized through homo-oligomerization of the centriolar protein SAS-6, but whether SAS-6 self-assembly can dictate cartwheel and thereby centriole symmetry is unclear. Here we show that Leishmania major SAS-6 crystallizes as a 9-fold symmetric cartwheel and provide the X-ray structure of this assembly at a resolution of 3.5 Å. We furthermore demonstrate that oligomerization of Leishmania SAS-6 can be inhibited by a small molecule in vitro and provide indications for its binding site. Our results firmly establish that SAS-6 can impose cartwheel symmetry on its own and indicate how this process might occur mechanistically in vivo. Importantly, our data also provide a proof-of-principle that inhibition of SAS-6 oligomerization by small molecules is feasible. DOI: http://dx.doi.org/10.7554/eLife.01812.001

Structural basis for Pan3 binding to Pan2 and its function in mRNA recruitment and deadenylation

The conserved eukaryotic Pan2–Pan3 deadenylation complex shortens cytoplasmic mRNA 3′ polyA tails to regulate mRNA stability. Although the exonuclease activity resides in Pan2, efficient deadenylation requires Pan3. The mechanistic role of Pan3 is unclear. Here, we show that Pan3 binds RNA directly both through its pseudokinase/C‐terminal domain and via an N‐terminal zinc finger that binds polyA RNA specifically. In contrast, isolated Pan2 is unable to bind RNA. Pan3 binds to the region of Pan2 that links its N‐terminal WD40 domain to the C‐terminal part that contains the exonuclease, with a 2:1 stoichiometry. The crystal structure of the Pan2 linker region bound to a Pan3 homodimer shows how the unusual structural asymmetry of the Pan3 dimer is used to form an extensive high‐affinity interaction. This binding allows Pan3 to supply Pan2 with substrate polyA RNA, facilitating efficient mRNA deadenylation by the intact Pan2–Pan3 complex.

Folding and stability of the ligand-binding domain of the glucocorticoid receptor.

A complex pathway involving many molecular chaperones has been proposed for the folding, assembly, and maintenance of a high‐affinity ligand‐binding form of steroid receptors in vivo, including the glucocorticoid receptor. To better understand this intricate folding and assembly process, we studied the folding of the ligand‐binding domain of the glucocorticoid receptor in vitro. We found that this domain can be refolded into a compact, highly structured state in vitro in the absence of chaperones. However, the presence of zwitterionic detergent is required to maintain the domain in a soluble form. In this state, the protein is dimeric and has considerable helical structure as shown by far‐UV circular dichroism. Further investigation of the properties of this in vitro refolded state show that it is stable and resistant to denaturation by heat or low concentrations of chemical denaturants. A detailed analysis of the unfolding equilibria using three different structural probes demonstrated that this state unfolds via a highly populated dimeric intermediate state. Together, these data clearly show that the ligand‐binding domain of the glucocorticoid receptor does not require chaperones for folding per se. However, this in vitro refolded state binds the ligand dexamethasone only weakly (Kd = 45 μM) compared to the in vivo assembled receptor (Kd = 3.4 nM). We suggest that the role of Hsp90 and associated chaperones is to bind to, and stabilize, a specific conformational state of the receptor which binds ligand with high affinity.

Structural Insights into Arl1-Mediated Targeting of the Arf-GEF BIG1 to the trans-Golgi

Summary The GTPase Arf1 is the major regulator of vesicle traffic at both the cis- and trans-Golgi. Arf1 is activated at the cis-Golgi by the guanine nucleotide exchange factor (GEF) GBF1 and at the trans-Golgi by the related GEF BIG1 or its paralog, BIG2. The trans-Golgi-specific targeting of BIG1 and BIG2 depends on the Arf-like GTPase Arl1. We find that Arl1 binds to the dimerization and cyclophilin binding (DCB) domain in BIG1 and report a crystal structure of human Arl1 bound to this domain. Residues in the DCB domain that bind Arl1 are required for BIG1 to locate to the Golgi in vivo. DCB domain-binding residues in Arl1 have a distinct conformation from those in known Arl1-effector complexes, and this plasticity allows Arl1 to interact with different effectors of unrelated structure. The findings provide structural insight into how Arf1 GEFs, and hence active Arf1, achieve their correct subcellular distribution.