Self‐association of TPR domains: Lessons learned from a designed,consensus‐based TPR oligomer

The tetratricopeptide repeat (TPR) motif is a protein–protein interaction module that acts as an organizing centre for complexes regulating a multitude of biological processes. Despite accumulating evidence for the formation of TPR oligomers as an additional level of regulation there is a lack of structural and solution data explaining TPR self‐association. In the present work we characterize the trimeric TPR‐containing protein YbgF, which is linked to the Tol system in Gram‐negative bacteria. By subtracting previously identified TPR consensus residues required for stability of the fold from residues conserved across YbgF homologs, we identified residues involved in oligomerization of the C‐terminal YbgF TPR domain. Crafting these residues, which are located in loop regions between TPR motifs, onto the monomeric consensus TPR protein CTPR3 induced the formation of oligomers. The crystal structure of this engineered oligomer shows an asymmetric trimer where stacking interactions between the introduced tyrosines and displacement of the C‐terminal hydrophilic capping helix, present in most TPR domains, are key to oligomerization. Asymmetric trimerization of the YbgF TPR domain and CTPR3Y3 leads to the formation of higher order oligomers both in the crystal and in solution. However, such open‐ended self‐association does not occur in full‐length YbgF suggesting that the protein's N‐terminal coiled‐coil domain restricts further oligomerization. This interpretation is borne out in experiments where the coiled‐coil domain of YbgF was engineered onto the N‐terminus of CTPR3Y3 and shown to block self‐association beyond trimerization. Our study lays the foundations for understanding the structural basis for TPR domain self‐association and how such self‐association can be regulated in TPR domain‐containing proteins. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Structural mapping of the coiled‐coil domain of a bacterial condensin and comparative analyses across all domains of life suggest conserved features of SMC proteins

The structural maintenance of chromosomes (SMC) proteins form the cores of multisubunit complexes that are required for the segregation and global organization of chromosomes in all domains of life. These proteins share a common domain structure in which N‐ and C‐ terminal regions pack against one another to form a globular ATPase domain. This “head” domain is connected to a central, globular, “hinge” or dimerization domain by a long, antiparallel coiled coil. To date, most efforts for structural characterization of SMC proteins have focused on the globular domains. Recently, however, we developed a method to map interstrand interactions in the 50‐nm coiled‐coil domain of MukB, the divergent SMC protein found in γ‐proteobacteria. Here, we apply that technique to map the structure of the Bacillus subtilis SMC (BsSMC) coiled‐coil domain. We find that, in contrast to the relatively complicated coiled‐coil domain of MukB, the BsSMC domain is nearly continuous, with only two detectable coiled‐coil interruptions. Near the middle of the domain is a break in coiled‐coil structure in which there are three more residues on the C‐terminal strand than on the N‐terminal strand. Close to the head domain, there is a second break with a significantly longer insertion on the same strand. These results provide an experience base that allows an informed interpretation of the output of coiled‐coil prediction algorithms for this family of proteins. A comparison of such predictions suggests that these coiled‐coil deviations are highly conserved across SMC types in a wide variety of organisms, including humans. Proteins 2015; 83:1027–1045. © 2015 Wiley Periodicals, Inc.     

BECN2 interacts with ATG14 through a metastable coiled‐coil to mediate autophagy

ATG14 binding to BECN/Beclin homologs is essential for autophagy, a critical catabolic homeostasis pathway. Here, we show that the α‐helical, coiled‐coil domain (CCD) of BECN2, a recently identified mammalian BECN1 paralog, forms an antiparallel, curved homodimer with seven pairs of nonideal packing interactions, while the BECN2 CCD and ATG14 CCD form a parallel, curved heterodimer stabilized by multiple, conserved polar interactions. Compared to BECN1, the BECN2 CCD forms a weaker homodimer, but binds more tightly to the ATG14 CCD. Mutation of nonideal BECN2 interface residues to more ideal pairs improves homodimer self‐association and thermal stability. Unlike BECN1, all BECN2 CCD mutants bind ATG14, although more weakly than wild type. Thus, polar BECN2 CCD interface residues result in a metastable homodimer, facilitating dissociation, but enable better interactions with polar ATG14 residues stabilizing the BECN2:ATG14 heterodimer. These structure‐based mechanistic differences in BECN1 and BECN2 homodimerization and heterodimerization likely dictate competitive ATG14 recruitment.     

Structure of the N‐terminal domain of the metalloprotease PrtV from Vibrio cholerae

The metalloprotease PrtV from Vibrio cholerae serves an important function for the ability of bacteria to invade the mammalian host cell. The protein belongs to the family of M6 proteases, with a characteristic zinc ion in the catalytic active site. PrtV constitutes a 918 amino acids (102 kDa) multidomain pre‐pro‐protein that undergoes several N‐ and C‐terminal modifications to form a catalytically active protease. We report here the NMR structure of the PrtV N‐terminal domain (residues 23–103) that contains two short α‐helices in a coiled coil motif. The helices are held together by a cluster of hydrophobic residues. Approximately 30 residues at the C‐terminal end, which were predicted to form a third helical structure, are disordered. These residues are highly conserved within the genus Vibrio, which suggests that they might be functionally important.     

Coiled‐coils: The long and short of it

Coiled‐coils are found in proteins throughout all three kingdoms of life. Coiled‐coil domains of some proteins are almost invariant in sequence and length, betraying a structural and functional role for amino acids along the entire length of the coiled‐coil. Other coiled‐coils are divergent in sequence, but conserved in length, thereby functioning as molecular spacers. In this capacity, coiled‐coil proteins influence the architecture of organelles such as centrioles and the Golgi, as well as permit the tethering of transport vesicles. Specialized coiled‐coils, such as those found in motor proteins, are capable of propagating conformational changes along their length that regulate cargo binding and motor processivity. Coiled‐coil domains have also been identified in enzymes, where they function as molecular rulers, positioning catalytic activities at fixed distances. Finally, while coiled‐coils have been extensively discussed for their potential to nucleate and scaffold large macromolecular complexes, structural evidence to substantiate this claim is relatively scarce.     

Identification of the amino acid residues and domains in the cysteine‐rich protein of Chinese wheat mosaic virus that are important for RNA silencing suppression and subcellular localization

Cysteine‐rich proteins (CRPs) encoded by some plant viruses in diverse genera function as RNA silencing suppressors. Within the N‐terminal portion of CRPs encoded by furoviruses, there are six conserved cysteine residues and a Cys–Gly–X–X–His motif (Cys, cysteine; Gly, glycine; His, histidine; X, any amino acid residue) with unknown function. The central domains contain coiled‐coil heptad amino acid repeats that usually mediate protein dimerization. Here, we present evidence that the conserved cysteine residues and Cys–Gly–X–X–His motif in the CRP of Chinese wheat mosaic virus (CWMV) are critical for protein stability and silencing suppression activity. Mutation of a leucine residue in the third coiled‐coil heptad impaired CWMV CRP activity for suppression of local silencing, but not for the promotion of cell‐to‐cell movement of Potato virus X (PVX). In planta and in vitro analysis of wild‐type and mutant proteins indicated that the ability of the CRP to self‐interact was correlated with its suppression activity. Deletion of up to 40 amino acids at the C‐terminus did not abolish suppression activity, but disrupted the association of CRP with endoplasmic reticulum (ER), and reduced its activity in the enhancement of PVX symptom severity. Interestingly, a short region in the C‐terminal domain, predicted to form an amphipathic α‐helical structure, was responsible for the association of CWMV CRP with ER. Overall, our results demonstrate that the N‐terminal and central regions are the functional domains for suppression activity, whereas the C‐terminal region primarily functions to target CWMV CRP to the ER.     

The DNA‐ and RNA‐binding protein FACTOR of DNA METHYLATION 1 requires XH domain‐mediated complex formation for its function in RNA‐directed DNA methylation

Studies have identified a sub‐group of SGS3‐LIKE proteins including FDM1–5 and IDN2 as key components of RNA‐directed DNA methylation pathway (RdDM). Although FDM1 and IDN2 bind RNAs with 5′ overhangs, their functions in the RdDM pathway remain to be examined. Here we show that FDM1 interacts with itself and with IDN2. Gel filtration suggests that FDM1 may exist as a homodimer in a heterotetramer complex in vivo. The XH domain of FDM1 mediates the FDM1–FDM1 and FDM1–IDN2 interactions. Deletion of the XH domain disrupts FDM1 complex formation and results in loss‐of‐function of FDM1. These results demonstrate that XH domain‐mediated complex formation of FDM1 is required for its function in RdDM. In addition, FDM1 binds unmethylated but not methylated DNAs through its coiled‐coil domain. RNAs with 5′ overhangs does not compete with DNA for binding by FDM1, indicating that FDM1 may bind DNA and RNA simultaneously. These results provide insight into how FDM1 functions in RdDM.     

Mapping of the chaperone AcrH binding regions of translocators AopB and AopD and characterization of oligomeric and metastable AcrH‐AopB‐AopD complexes in the type III secretion system of Aeromonas hydrophila

In the type III secretion system (T3SS) of Aeromonas hydrophila, AcrH acts as a chaperone for translocators AopB and AopD. AcrH forms a stable 1:1 monomeric complex with AopD, whereas the 1:1 AcrH‐AopB complex exists mainly as a metastable oligomeric form and only in minor amounts as a stable monomeric form. Limited protease digestion shows that these complexes contain highly exposed regions, thus allowing mapping of intact functional chaperone binding regions of AopB and AopD. AopD uses the transmembrane domain (DF1, residues 16–147) and the C‐terminal amphipathic helical domain (DF2, residues 242–296) whereas AopB uses a discrete region containing the transmembrane domain and the putative N‐terminal coiled coil domain (BF1, residues 33–264). Oligomerization of the AcrH‐AopB complex is mainly through the C‐terminal coiled coil domain of AopB, which is dispensable for chaperone binding. The three proteins, AcrH, AopB, and AopD, can be coexpressed to form an oligomeric and metastable complex. These three proteins are also oligomerized mainly through the C‐terminal domain of AopB. Formation of such an oligomeric and metastable complex may be important for the proper formation of translocon of correct topology and stoichiometry on the host membrane.     

Crystal structure of a super leucine zipper,an extended two‐stranded super long coiled coil

Coiled coil is a ubiquitous structural motif in proteins, with two to seven alpha helices coiled together like the strands of a rope, and coiled coil folding and assembly is not completely understood. A GCN4 leucine zipper mutant with four mutations of K3A, D7A, Y17W, and H18N has been designed, and the crystal structure has been determined at 1.6 Å resolution. The peptide monomer shows a helix trunk with short curved N‐ and C‐termini. In the crystal, two monomers cross in 35° and form an X‐shaped dimer, and each X‐shaped dimer is welded into the next one through sticky hydrophobic ends, thus forming an extended two‐stranded, parallel, super long coiled coil rather than a discrete, two‐helix coiled coil of the wild‐type GCN4 leucine zipper. Leucine residues appear at every seventh position in the super long coiled coil, suggesting that it is an extended super leucine zipper. Compared to the wild‐type leucine zipper, the N‐terminus of the mutant has a dramatic conformational change and the C‐terminus has one more residue Glu 32 determined. The mutant X‐shaped dimer has a large crossing angle of 35° instead of 18° in the wild‐type dimer. The results show a novel assembly mode and oligomeric state of coiled coil, and demonstrate that mutations may affect folding and assembly of the overall coiled coil. Analysis of the formation mechanism of the super long coiled coil may help understand and design self‐assembling protein fibers.     

X‐ray crystal structure of the N‐terminal region of Moloney murine leukemia virus integrase and its implications for viral DNA recognition

The retroviral integrase (IN) carries out the integration of a dsDNA copy of the viral genome into the host DNA, an essential step for viral replication. All IN proteins have three general domains, the N‐terminal domain (NTD), the catalytic core domain, and the C‐terminal domain. The NTD includes an HHCC zinc finger‐like motif, which is conserved in all retroviral IN proteins. Two crystal structures of Moloney murine leukemia virus (M‐MuLV) IN N‐terminal region (NTR) constructs that both include an N‐terminal extension domain (NED, residues 1–44) and an HHCC zinc‐finger NTD (residues 45–105), in two crystal forms are reported. The structures of IN NTR constructs encoding residues 1–105 (NTR_1–105) and 8–105 (NTR_8–105) were determined at 2.7 and 2.15 Å resolution, respectively and belong to different space groups. While both crystal forms have similar protomer structures, NTR_1–105 packs as a dimer and NTR_8–105 packs as a tetramer in the asymmetric unit. The structure of the NED consists of three anti‐parallel β‐strands and an α‐helix, similar to the NED of prototype foamy virus (PFV) IN. These three β‐strands form an extended β‐sheet with another β‐strand in the HHCC Zn²⁺ binding domain, which is a unique structural feature for the M‐MuLV IN. The HHCC Zn²⁺ binding domain structure is similar to that in HIV and PFV INs, with variations within the loop regions. Differences between the PFV and MLV IN NEDs localize at regions identified to interact with the PFV LTR and are compared with established biochemical and virological data for M‐MuLV. Proteins 2017; 85:647–656. © 2016 Wiley Periodicals, Inc.     

Influence of a heptad repeat stutter on the pH‐dependent conformational behavior of the central coiled‐coil from influenza hemagglutinin HA2

The coiled‐coil is one of the most common protein structural motifs. Amino acid sequences of regions that participate in coiled‐coils contain a heptad repeat in which every third then forth residue is occupied by a hydrophobic residue. Here we examine the consequences of a “stutter,” a deviation of the idealized heptad repeat that is found in the central coiled‐coil of influenza hemagluttinin HA2. Characterization of a peptide containing the native stutter‐containing HA2 sequence, as well as several variants in which the stutter was engineered out to restore an idealized heptad repeat pattern, revealed that the stutter is important for allowing coiled‐coil formation in the WT HA2 at both neutral and low pH (7.1 and 4.5). By contrast, all variants that contained idealized heptad repeats exhibited marked pH‐dependent coiled‐coil formation with structures forming much more stably at low pH. A crystal structure of one variant containing an idealized heptad repeat, and comparison to the WT HA2 structure, suggest that the stutter distorts the optimal interhelical core packing arrangement, resulting in unwinding of the coiled‐coil superhelix. Interactions between acidic side chains, in particular E69 and E74 (present in all peptides studied), are suggested to play a role in mediating these pH‐dependent conformational effects. This conclusion is partially supported by studies on HA2 variant peptides in which these positions were altered to aspartic acid. These results provide new insight into the structural role of the heptad repeat stutter in HA2. Proteins 2014; 82:2220–2228. © 2014 Wiley Periodicals, Inc.     

The PHCCEx domain of Tiam1/2 is a novel protein‐ and membrane‐binding module

Tiam1 and Tiam2 (Tiam1/2) are guanine nucleotide‐exchange factors that possess the PH–CC–Ex (pleckstrin homology, coiled coil and extra) region that mediates binding to plasma membranes and signalling proteins in the activation of Rac GTPases. Crystal structures of the PH–CC–Ex regions revealed a single globular domain, PHCCEx domain, comprising a conventional PH subdomain associated with an antiparallel coiled coil of CC subdomain and a novel three‐helical globular Ex subdomain. The PH subdomain resembles the β‐spectrin PH domain, suggesting non‐canonical phosphatidylinositol binding. Mutational and binding studies indicated that CC and Ex subdomains form a positively charged surface for protein binding. We identified two unique acidic sequence motifs in Tiam1/2‐interacting proteins for binding to PHCCEx domain, Motif‐I in CD44 and ephrinB's and the NMDA receptor, and Motif‐II in Par3 and JIP2. Our results suggest the molecular basis by which the Tiam1/2 PHCCEx domain facilitates dual binding to membranes and signalling proteins.     

Structure of dystrophia myotonica protein kinase

Dystrophia myotonica protein kinase (DMPK) is a serine/threonine kinase composed of a kinase domain and a coiled‐coil domain involved in the multimerization. The crystal structure of the kinase domain of DMPK bound to the inhibitor bisindolylmaleimide VIII (BIM‐8) revealed a dimeric enzyme associated by a conserved dimerization domain. The affinity of dimerisation suggested that the kinase domain alone is insufficient for dimerisation in vivo and that the coiled‐coil domains are required for stable dimer formation. The kinase domain is in an active conformation, with a fully‐ordered and correctly positioned αC helix, and catalytic residues in a conformation competent for catalysis. The conserved hydrophobic motif at the C‐terminal extension of the kinase domain is bound to the N‐terminal lobe of the kinase domain, despite being unphosphorylated. Differences in the arrangement of the C‐terminal extension compared to the closely related Rho‐associated kinases include an altered PXXP motif, a different conformation and binding arrangement for the turn motif, and a different location for the conserved NFD motif. The BIM‐8 inhibitor occupies the ATP site and has similar binding mode as observed in PDK1.     

The Rise of the Cartwheel: Seeding the Centriole Organelle

The cartwheel is a striking structure critical for building the centriole, a microtubule‐based organelle fundamental for organizing centrosomes, cilia, and flagella. Over the last 50 years, the cartwheel has been described in many systems using electron microscopy, but the molecular nature of its constituent building blocks and their assembly mechanisms have long remained mysterious. Here, we review discoveries that led to the current understanding of cartwheel structure, assembly, and function. We focus on the key role of SAS‐6 protein self‐organization, both for building the signature ring‐like structure with hub and spokes, as well as for their vertical stacking. The resemblance of assembly intermediates in vitro and in vivo leads us to propose a novel registration step in cartwheel biogenesis, whereby stacked SAS‐6‐containing rings are put in register through interactions with peripheral elements anchored to microtubules. We conclude by evoking some avenues for further uncovering cartwheel and centriole assembly mechanisms.     

Instability in a coiled‐coil signaling helix is conserved for signal transduction in soluble guanylyl cyclase

How nitric oxide (NO) activates its primary receptor, α1/β1 soluble guanylyl cyclase (sGC or GC‐1), remains unknown. Likewise, how stimulatory compounds enhance sGC activity is poorly understood, hampering development of new treatments for cardiovascular disease. NO binding to ferrous heme near the N‐terminus in sGC activates cyclase activity near the C‐terminus, yielding cGMP production and physiological response. CO binding can also stimulate sGC, but only weakly in the absence of stimulatory small‐molecule compounds, which together lead to full activation. How ligand binding enhances catalysis, however, has yet to be discovered. Here, using a truncated version of sGC from Manduca sexta, we demonstrate that the central coiled‐coil domain, the most highly conserved region of the ~150,000 Da protein, not only provides stability to the heterodimer but is also conformationally active in signal transduction. Sequence conservation in the coiled coil includes the expected heptad‐repeating pattern for coiled‐coil motifs, but also invariant positions that disfavor coiled‐coil stability. Full‐length coiled coil dampens CO affinity for heme, while shortening of the coiled coil leads to enhanced CO binding. Introducing double mutation αE447L/βE377L, predicted to replace two destabilizing glutamates with leucines, lowers CO binding affinity while increasing overall protein stability. Likewise, introduction of a disulfide bond into the coiled coil results in reduced CO affinity. Taken together, we demonstrate that the heme domain is greatly influenced by coiled‐coil conformation, suggesting communication between heme and catalytic domains is through the coiled coil. Highly conserved structural imperfections in the coiled coil provide needed flexibility for signal transduction.     

Features critical for membrane binding revealed by DivIVA crystal structure

DivIVA is a conserved protein in Gram‐positive bacteria that localizes at the poles and division sites, presumably through direct sensing of membrane curvature. DivIVA functions as a scaffold and is vital for septum site selection during vegetative growth and chromosome anchoring during sporulation. DivIVA deletion causes filamentous growth in Bacillus subtilis, whereas overexpression causes hyphal branching in Streptomyces coelicolor. We have determined the crystal structure of the N‐terminal (Nt) domain of DivIVA, and show that it forms a parallel coiled‐coil. It is capped with two unique crossed and intertwined loops, exposing hydrophobic and positively charged residues that we show here are essential for membrane binding. An intragenic suppressor introducing a positive charge restores membrane binding after mutating the hydrophobic residues. We propose that the hydrophobic residues insert into the membrane and that the positively charged residues bind to the membrane surface. A low‐resolution crystal structure of the C‐terminal (Ct) domain displays a curved tetramer made from two parallel coiled‐coils. The Nt and Ct parts were then merged into a model of the full length, 30 nm long DivIVA protein.