Scaffolding proteins and their role in viral assembly

Scaffolding proteins are proteins that are required to catalyse, regulate or modulate some step in the assembly of a macromolecular complex. They associate specifically with the nascent protein complex during assembly, but are subsequently removed, and are absent from the mature structure. Scaffolding proteins have been described primarily from viral systems, in particular from the double-stranded DNA bacteriophages, but most likely play a more general role in macromolecular assembly, a fundamental process in all biological systems. Scaffolding proteins may act in a specific fashion, by actively encouraging the formation of correct protein-protein interactions, or more generally by nucleating and promoting assembly. They may also work to ensure the fidelity of the assembly process by preventing the formation of improper interactions, in many ways similar to the role of molecular chaperones in protein folding. In viruses, scaffolding proteins are found both in the form of internal cores and external bracing, and may form elaborate and complex structures. This review will focus on the viral scaffolding proteins, for which an increasing amount of structural and functional information has recently become available.     

RNA recognition by double-stranded RNA binding domains: a matter of shape and sequence

The double-stranded RNA binding domain (dsRBD) is a small protein domain of 65–70 amino acids adopting an αβββα fold, whose central property is to bind to double-stranded RNA (dsRNA). This domain is present in proteins implicated in many aspects of cellular life, including antiviral response, RNA editing, RNA processing, RNA transport and, last but not least, RNA silencing. Even though proteins containing dsRBDs can bind to very specific dsRNA targets in vivo, the binding of dsRBDs to dsRNA is commonly believed to be shape-dependent rather than sequence-specific. Interestingly, recent structural information on dsRNA recognition by dsRBDs opens the possibility that this domain performs a direct readout of RNA sequence in the minor groove, allowing a global reconsideration of the principles describing dsRNA recognition by dsRBDs. We review in this article the current structural and molecular knowledge on dsRBDs, emphasizing the intricate relationship between the amino acid sequence, the structure of the domain and its RNA recognition capacity. We especially focus on the molecular determinants of dsRNA recognition and describe how sequence discrimination can be achieved by this type of domain.     

Kinesin-II, the heteromeric kinesin

The kinesins constitute a large family of motor proteins which are responsible for the distribution of numerous organelles, vesicles and macromolecular complexes throughout the cell. One class of these molecular motors, kinesin-II, is unique in that these proteins are typically found as heterotrimeric complexes containing two different, though related, kinesin-like motor subunits, and a single nonmotor subunit. The heteromeric nature of these kinesins appears to have resulted in a class of combinatorial kinesins which can 'mix and match' different motor subunits. Another novel feature of these motors is that the activities of several kinesin-II representatives are essential in the assembly of motile and nonmotile cilia, a role not attributed to any other kinesin. This review presents a brief overview of the structure and biological functions of kinesin-II, the heteromeric kinesin.     

Viral capsids: Mechanical characteristics,genome packaging and delivery mechanisms

The main functions of viral capsids are to protect, transport and deliver their genome. The mechanical properties of capsids are supposed to be adapted to these tasks. Bacteriophage capsids also need to withstand the high pressures the DNA is exerting onto it as a result of the DNA packaging and its consequent confinement within the capsid. It is proposed that this pressure helps driving the genome into the host, but other mechanisms also seem to play an important role in ejection. DNA packaging and ejection strategies are obviously dependent on the mechanical properties of the capsid. This review focuses on the mechanical properties of viral capsids in general and the elucidation of the biophysical aspects of genome packaging mechanisms and genome delivery processes of double-stranded DNA bacteriophages in particular. Received 14 October 2006; received after revision 18 December 2006; accepted 27 February 2007     

The bacterial translocase: a dynamic protein channel complex

The major route of protein translocation in bacteria is the so-called general secretion pathway (Sec-pathway). This route has been extensively studied in Escherichia coli and other bacteria. The movement of preproteins across the cytoplasmic membrane is mediated by a multimeric membrane protein complex called translocase. The core of the translocase consists of a proteinaceous channel formed by an oligomeric assembly of the heterotrimeric membrane protein complex SecYEG and the peripheral adenosine triphosphatase (ATPase) SecA as molecular motor. Many secretory proteins utilize the molecular chaperone SecB for targeting and stabilization of the unfolded state prior to translocation, while most nascent inner membrane proteins are targeted to the translocase by the signal recognition particle and its membrane receptor. Translocation is driven by ATP hydrolysis and the proton motive force. In the last decade, genetic and biochemical studies have provided detailed insights into the mechanism of preprotein translocation. Recent crystallographic studies on SecA, SecB and the SecYEG complex now provide knowledge about the structural features of the translocation process. Here, we will discuss the mechanistic and structural basis of the translocation of proteins across and the integration of membrane proteins into the cytoplasmic membrane.Received 10 January 2003; received after revision 2 April 2003; accepted 4 April 2003     

Bidirectional motility of kinesin-5 motor proteins: structural determinants,cumulative functions and physiological roles

Mitotic kinesin-5 bipolar motor proteins perform essential functions in mitotic spindle dynamics by crosslinking and sliding antiparallel microtubules (MTs) apart within the mitotic spindle. Two recent studies have indicated that single molecules of Cin8, the Saccharomyces cerevisiae kinesin-5 homolog, are minus end-directed when moving on single MTs, yet switch directionality under certain experimental conditions (Gerson-Gurwitz et al., EMBO J 30:4942–4954, 2011; Roostalu et al., Science 332:94–99, 2011). This finding was unexpected since the Cin8 catalytic motor domain is located at the N-terminus of the protein, and such kinesins have been previously thought to be exclusively plus end-directed. In addition, the essential intracellular functions of kinesin-5 motors in separating spindle poles during mitosis can only be accomplished by plus end-directed motility during antiparallel sliding of the spindle MTs. Thus, the mechanism and possible physiological role of the minus end-directed motility of kinesin-5 motors remain unclear. Experimental and theoretical studies from several laboratories in recent years have identified additional kinesin-5 motors that are bidirectional, revealed structural determinants that regulate directionality, examined the possible mechanisms involved and have proposed physiological roles for the minus end-directed motility of kinesin-5 motors. Here, we summarize our current understanding of the remarkable ability of certain kinesin-5 motors to switch directionality when moving along MTs.     

Role of dystrophin and utrophin for assembly and function of the dystrophin glycoprotein complex in non-muscle tissue

The dystrophin glycoprotein complex (DGC) is a multimeric protein assembly associated with either the X-linked cytoskeletal protein dystrophin or its autosomal homologue utrophin. In striated muscle cells, the DGC links the extracellular matrix to the actin cytoskeleton and mediates three major functions: structural stability of the plasma membrane, ion homeostasis, and transmembrane signaling. Mutations affecting the DGC underlie major forms of congenital muscle dystrophies. The DGC is prominent also in the central and peripheral nervous system and in tissues with a secretory function or which form barriers between functional compartments, such as the blood-brain barrier, choroid plexus, or kidney. A considerable molecular heterogeneity arises from cell-specific expression of its constituent proteins, notably short C-terminal isoforms of dystrophin. Experimentally, the generation of mice carrying targeted gene deletions affecting the DGC has clarified the interdependence of DGC proteins for assembly of the complex and revealed its importance for brain development and regulation of the ’milieu intérieur. Here, we focus on recent studies of the DGC in brain, blood-brain barrier and choroid plexus, retina, and kidney and discuss the role of dystrophin isoforms and utrophin for assembly of the complex in these tissues. Received 4 October 2005; received after revision 14 March 2006; accepted 5 April 2006     

Structure and dynamics of rotary V<Subscript>1</Subscript> motor

Rotary ATPases are unique rotary molecular motors that function as energy conversion machines. Among all known rotary ATPases, F₁-ATPase is the best characterized rotary molecular motor. There are many high-resolution crystal structures and the rotation dynamics have been investigated in detail by extensive single-molecule studies. In contrast, knowledge on the structure and rotation dynamics of V₁-ATPase, another rotary ATPase, has been limited. However, recent high-resolution structural studies and single-molecule studies on V₁-ATPase have provided new insights on how the catalytic sites in this molecular motor change its conformation during rotation driven by ATP hydrolysis. In this review, we summarize recent information on the structural features and rotary dynamics of V₁-ATPase revealed from structural and single-molecule approaches and discuss the possible chemomechanical coupling scheme of V₁-ATPase with a focus on differences between rotary molecular motors.     

Biogenesis of mitochondrial porin: the import pathway.

We review here the present knowledge about the pathway of import and assembly of porin into mitochondria and compare it to those of other mitochondrial proteins. Porin, like all outer mitochondrial membrane proteins studied so far is made as a precursor without a cleavable 'signal' sequence; thus targeting information must reside in the mature sequence. At least part of this information appears to be located at the amino-terminal end of the molecule. Transport into mitochondria can occur post-translationally. In a first step, the porin precursor is specifically recognized on the mitochondrial surface by a protease sensitive receptor. In a second step, porin precursor inserts partially into the outer membrane. This step is mediated by a component of the import machinery common to the import pathways of precursor proteins destined for other mitochondrial subcompartments. Finally, porin is assembled to produce the functional oligomeric form of an integral membrane protein which is characterized by its extreme protease resistance.     

Breaking biological symmetry in membrane proteins: The asymmetrical orientation of PsaC on the pseudo-C2 symmetric Photosystem I core

The elucidation of assembly pathways of multi-subunit membrane proteins is of growing interest in structural biology. In this study, we provide an analysis of the assembly of the asymmetrically oriented PsaC subunit on the pseudo C₂-symmetric Photosystem I core. Based on a comparison of the differences in the NMR solution structure of unbound PsaC with that of the X-ray crystal structure of bound PsaC, and on a detailed analysis of the PsaC binding site surrounding the F_X iron-sulfur cluster, two models can be envisioned for what are likely the last steps in the assembly of Photosystem I. Here, we dissect both models and attempt to address heretofore unrecognized issues by proposing a mechanism that includes a thermodynamic perspective. Experimental strategies to verify the models are proposed. In closing, the evolutionary aspects of the assembly process will be considered, with special reference to the structural arrangement of the PsaC binding surface. Received 22 October 2008; received after revision 17 November 2008; accepted 05 December 2008     

P58IPK, a novel cochaperone containing tetratricopeptide repeats and a J-domain with oncogenic potential

Tetratricopeptide repeats (TPRs) are loosely conserved 34-amino acid sequence motifs that have been shown to function as scaffolding structures to mediate protein-protein interactions. TPRs have been identified in a number of proteins with diverse functions and cellular locations. Recent studies suggest that individual TPR motifs can confer specificity in promoting homotypic and/or heterotypic interactions, often in a mutually exclusive manner. These features are best exemplified by the P58IPK protein, an influenza virus-activated cellular inhibitor of the PKR protein kinase, whose different TPR motifs mediate interactions with distinct proteins. P58IPK, which possesses cochaperone and oncogenic properties, represents a unique class of TPR proteins containing a J-domain. Here we review recent progress on the structural and functional characterization of P58IPK, and discuss the possible mechanisms by which P58IPK modulates PKR and induces tumorigenesis in view of present knowledge of TPR proteins and molecular chaperones.     

Assembly and genetics of spore protective structures

The sporulation program in Bacillus subtilis ends in the formation of a highly resistant endospore that can withstand extremes of heat, mechanical disruption, ultraviolet irradiation, lytic enzymes and chemical attack. These properties are attributed mainly to the unique structure of spore coat and cortex, as well as to the physical state of the spore cytoplasm. The outermost layer of the spore, called the coat, has two morphologically distinct sublayers: an electron-dense outer coat and an electron-translucent inner coat. The coat is composed of more than 2 dozen proteins of varying size. Many coat genes and coat proteins have been isolated and characterized in detail, and studies of these have identified proteins with important roles in coat assembly, resistance and spore germination. We describe here characteristics of the coat proteins and propose a model for coat assembly based on recent work.     

2’-5’ Oligoadenylate synthetase shares active site architecture with the archaeal CCA-adding enzyme

The 2'-5' oligoadenylate synthetases (OAS) are interferon-induced antiviral enzymes that recognise virally produced dsRNA and initiate an RNA destabilisation within the infected cell. We compared the structure of OAS to that of poly adenosine polymerase (PAP) and the class I CCA-adding enzyme from Archeoglobus fulgidus (AfCCA). This comparison revealed a strong structural homology between the three enzyme families. In particular, the active sites of OAS and CCA class I enzymes are highly conserved. We conducted an extensive mutagenesis of amino acid residues within the putative active site in OAS, thereby identifying enzymatically important residues and confirming the common active site architecture for OAS and the AfCCA. Our findings also have profound implications for our understanding of the evolutionary origin of the OAS enzymes and suggest that the OAS proteins diverged from a common 3'-specific ancestor at the beginning of metazoan evolution.     

Structure and function of desmosomal proteins and their role in development and disease

Desmosomes represent major intercellular adhesive junctions at basolateral membranes of epithelial cells and in other tissues. They mediate direct cell-cell contacts and provide anchorage sites for intermediate filaments important for the maintenance of tissue architecture. There is increasing evidence now that desmosomes in addition to a simple structural function have new roles in tissue morphogenesis and differentiation. Transmembrane glycoproteins of the cadherin superfamily of Ca²⁺-dependent cell-cell adhesion molecules which mediate direct intercellular interactions in desmosomes appear to be of central importance in this respect. The complex network of proteins forming the desmosomal plaque associated with the cytoplasmic domain of the desmosomal cadherins, however, is also involved in junction assembly and regulation of adhesive strength. This re-view summarizes the structural features of these desmosomal proteins, their function during desmosome assembly and maintenance, and their role in development and disease.Received 5 February 2003; received after revision 14 March 2003; accepted 1 April 2003     

Biogenesis of mitochondrial porin: The import pathway

Summary We review here the present knowledge about the pathway of import and assembly of porin into mitochondria and compare it to those of other mitochondrial proteins. Porin, like all outer mitochondrial membrane proteins studied so far is made as a precursor without a cleavble signal sequence; thus targeting information must reside in the mature sequence. At least part of this information appears to be located at the amino-terminal end of the molecule. Transport into mitochondria can occur post-translationally. In a first step, the porin precursor is specifically recognized on the mitochondrial surface by a protease sensitive receptor. In a second step, porin precursor inserts partially into the outer membrane. This step is mediated by a component of the import machinery common to the import pathways of precursor proteins destined for other mitochondrial subcompartments. Finally, porin is assembled to produce the functional oligomeric form of an integral membrane protein wich is characterized by its extreme protease resistance.     

The role of the <Emphasis Type="Italic">trans</Emphasis>-Golgi network in varicella zoster virus biology

The task of assembling nascent virions presents a formidable challenge to large, enveloped DNA viruses such as varicella zoster virus (VZV). After parasitising the host cells compartmentalised biosynthetic machinery, viral constituents must be brought together in appropriate proportions for packaging and export. Recent evidence places the trans-Golgi network (TGN) in an orchestrating role with respect to the assembly, envelopment and egress of herpesviruses. This role accords with known functions of the TGN in the uninfected cell. The targeting of viral glycoproteins to the TGN appears to provide a crucial platform for viral assembly. Tegument proteins, interacting with the cytoplasmic domains of glycoproteins, in turn recruit nucleocapsids to the developing supramolecular array. Molecular studies are continually refining understanding of these processes, building upon elegant electron microscopic data. Knowledge of VZVs use of endogenous trafficking pathways from the TGN sheds light on important aspects of viral behaviour in vitro and in vivo.Received 22 June 2004; received after revision 22 August 2004; accepted 25 August 2004     

Assessing heterogeneity in oligomeric AAA+ machines

ATPases Associated with various cellular Activities (AAA+ ATPases) are molecular motors that use the energy of ATP binding and hydrolysis to remodel their target macromolecules. The majority of these ATPases form ring-shaped hexamers in which the active sites are located at the interfaces between neighboring subunits. Structural changes initiate in an active site and propagate to distant motor parts that interface and reshape the target macromolecules, thereby performing mechanical work. During the functioning cycle, the AAA+ motor transits through multiple distinct states. Ring architecture and placement of the catalytic sites at the intersubunit interfaces allow for a unique level of coordination among subunits of the motor. This in turn results in conformational differences among subunits and overall asymmetry of the motor ring as it functions. To date, a large amount of structural information has been gathered for different AAA+ motors, but even for the most characterized of them only a few structural states are known and the full mechanistic cycle cannot be yet reconstructed. Therefore, the first part of this work will provide a broad overview of what arrangements of AAA+ subunits have been structurally observed focusing on diversity of ATPase oligomeric ensembles and heterogeneity within the ensembles. The second part of this review will concentrate on methods that assess structural and functional heterogeneity among subunits of AAA+ motors, thus bringing us closer to understanding the mechanism of these fascinating molecular motors.     

基于真实机器的装配公差分析方法

机器上目标要素的位置误差是零件几何误差和装配误差共同作用的结果．为了研究装配基准的几何误差在装配链中的传递和变换,提出基于真实机器模型的装配积累误差建模与分析方法．首先,采用控制点变动模型表示零件几何要素,利用蒙特卡罗模拟方法生成几何要素相对于公称位置的变动实例．然后,根据理论正确尺寸确定几何要素相对于基准参考框架的公称位置．第三,提出基准参考框架建立方法,根据基准要素实例确定目标要素的测量基准参考框架．最后,提出装配接触模型,根据装配顺序和基准要素位置实例确定测量基准参考框架相对于装配基准体系的位置．通过以上4个过程计算装配链中所有关联要素在机器坐标系下的位置,获得装配基准体系的几何误差在装配链中的转递和变换关系．将真实机器装配情况模拟分解为4个模块化过程,可以实现装配误差分析的自动化．以三平面定位的轴孔装配的装配间隙计算为实例,验证了本文提出的方法．