Microtubules pull the strings: disordered sequences as efficient couplers of microtubule-generated force

2020 ◽  
Vol 64 (2) ◽  
pp. 371-382
Author(s):  
Vladimir A. Volkov
Keyword(s):  
Binding Sites ◽  
Protein Complexes ◽  
Mechanical Tension ◽  
Microtubule Binding ◽  
Binding Domains ◽  
Force Coupling ◽  
Couple Dynamics ◽  
Systematic Understanding ◽  
Positively Charged ◽  
Cellular Organelles

Abstract Microtubules are dynamic polymers that grow and shrink through addition or loss of tubulin subunits at their ends. Microtubule ends generate mechanical force that moves chromosomes and cellular organelles, and provides mechanical tension. Recent literature describes a number of proteins and protein complexes that couple dynamics of microtubule ends to movements of their cellular cargoes. These ‘couplers’ are quite diverse in their microtubule-binding domains (MTBDs), while sharing similarity in function, but a systematic understanding of the principles underlying their activity is missing. Here, I review various types of microtubule couplers, focusing on their essential activities: ability to follow microtubule ends and capture microtubule-generated force. Most of the couplers require presence of unstructured positively charged sequences and multivalency in their microtubule-binding sites to efficiently convert the microtubule-generated force into useful connection to a cargo. An overview of the microtubule features supporting end-tracking and force-coupling, and the experimental methods to assess force-coupling properties is also provided.

scholarly journals Hsp90–Sgt1 and Skp1 target human Mis12 complexes to ensure efficient formation of kinetochore–microtubule binding sites

2010 ◽  
Vol 189 (2) ◽  
pp. 261-274 ◽  
Author(s):  
Alexander E. Davies ◽  
Kenneth B. Kaplan
Keyword(s):  
Binding Sites ◽  
Protein Complexes ◽  
Large Fraction ◽  
Multiprotein Complex ◽  
Complex Assembly ◽  
Microtubule Binding ◽  
Kinetochore Assembly ◽  
Chromosome Alignment ◽  
Chaperone Complex

The formation of functional kinetochores requires the accurate assembly of a large number of protein complexes. The Hsp90–Sgt1 chaperone complex is important for this process; however, its targets are not conserved and its exact contribution to kinetochore assembly is unclear. Here, we show that human Hsp90–Sgt1 interacts with the Mis12 complex, a so-called keystone complex required to assemble a large fraction of the kinetochore. Inhibition of Hsp90 or Sgt1 destabilizes the Mis12 complex and delays proper chromosome alignment due to inefficient formation of microtubule-binding sites. Interestingly, coinhibition of Sgt1 and the SCF subunit, Skp1, increases Mis12 complexes at kinetochores and restores timely chromosome alignment but forms less-robust microtubule-binding sites. We propose that a balance of Mis12 complex assembly and turnover is required for the efficient and accurate assembly of kinetochore–microtubule binding sites. These findings support a novel role for Hsp90–Sgt1 chaperones in ensuring the fidelity of multiprotein complex assembly.

scholarly journals Tetravalent single-chain avidin: from subunits to protein domains via circularly permuted avidins

2005 ◽  
Vol 392 (3) ◽  
pp. 485-491 ◽  
Author(s):  
Henri R. Nordlund ◽  
Vesa P. Hytönen ◽  
Jarno Hörhä ◽  
Juha A. E. Määttä ◽  
Daniel J. White ◽  
...  
Keyword(s):  
Binding Sites ◽  
Polypeptide Chain ◽  
Protein Complexes ◽  
Fusion Partner ◽  
Wild Type ◽  
Single Chain ◽  
Binding Domains ◽  
Single Polypeptide Chain ◽  
Biotin Binding ◽  
Single Polypeptide

scAvd (single-chain avidin, where two dcAvd are joined in a single polypeptide chain), having four biotin-binding domains, was constructed by fusion of topologically modified avidin units. scAvd showed similar biotin binding and thermal stability properties as chicken avidin. The DNA construct encoding scAvd contains four circularly permuted avidin domains, plus short linkers connecting the four domains into a single polypeptide chain. In contrast with wild-type avidin, which contains four identical avidin monomers, scAvd enables each one of the four avidin domains to be independently modified by protein engineering. Therefore the scAvd scaffold can be used to construct spatially and stoichiometrically defined pseudotetrameric avidin molecules showing different domain characteristics. In addition, unmodified scAvd could be used as a fusion partner, since it provides a unique non-oligomeric structure, which is fully functional with four high-affinity biotin-binding sites. Furthermore, the subunit-to-domain strategy described in the present study could be applied to other proteins and protein complexes, facilitating the development of sophisticated protein tools for applications in nanotechnology and life sciences.

Tuning of Electronic Properties in Conducting Polymers

2001 ◽  
Vol 66 (8) ◽  
pp. 1208-1218 ◽  
Author(s):  
Guofeng Li ◽  
Mira Josowicz ◽  
Jiří Janata
Keyword(s):  
Hydrogen Bonding ◽  
Binding Site ◽  
Binding Sites ◽  
Polymer Chains ◽  
Electronic Transitions ◽  
Weakly Bound ◽  
Ordered Structures ◽  
Doped Polymer ◽  
Positively Charged ◽  
Repeated Cycling

Structural and electronic transitions in poly(thiophenyleneiminophenylene), usually referred to as poly(phenylenesulfidephenyleneamine) (PPSA) upon electrochemical doping with LiClO4 have been investigated. The unusual electrochemical behavior of PPSA indicates that the dopant anions are bound in two energetically different sites. In the so-called "binding site", the ClO4- anion is Coulombically attracted to the positively charged S or N sites on one chain and simultaneously hydrogen-bonded with the N-H group on a neighboring polymer chain. This strong interaction causes a re-organization of the polymer chains, resulting in the formation of a networked structure linked together by these ClO4- Coulombic/hydrogen bonding "bridges". However, in the "non-binding site", the ClO4- anion is very weakly bound, involves only the electrostatic interaction and can be reversibly exchanged when the doped polymer is reduced. In the repeated cycling, the continuous and alternating influx and expulsion of ClO4- ions serves as a self-organizing process for such networked structures, giving rise to a diminishing number of available "non-binding" sites. The occurrence of these ordered structures has a major impact on the electrochemical activity and the morphology of the doped polymer. Also due to stabilization of the dopant ions, the doped polymer can be kept in a stable and desirable oxidation state, thus both work function and conductivity of the polymer can be electrochemically controlled.

Application of 2D BN/SDS-PAGE coupled with mass spectrometry for identification of VDAC-associated protein complexes related to mitochondrial binding sites for type I brain hexokinase

Mitochondrion ◽  
2013 ◽  
Vol 13 (6) ◽  
pp. 823-830 ◽  
Author(s):  
Carla Rossini Crepaldi ◽  
Phelipe Augusto Mariano Vitale ◽  
Andrea Cristina Tesch ◽  
Hélen Julie Laure ◽  
José César Rosa ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Binding Sites ◽  
Protein Complexes ◽  
Type I ◽  
Sds Page

scholarly journals Nuclear Import of TFIIB Is Mediated by Kap114p, a Karyopherin with Multiple Cargo-binding Domains

2005 ◽  
Vol 16 (7) ◽  
pp. 3200-3210 ◽  
Author(s):  
Jennifer L. Hodges ◽  
Jennifer H. Leslie ◽  
Nima Mosammaparast ◽  
Yurong Guo ◽  
Jeffrey Shabanowitz ◽  
...  
Keyword(s):  
Nuclear Import ◽  
Binding Sites ◽  
Binding Domains ◽  
Transcription Factor Iib ◽  
General Transcription Factor ◽  
Proteomic Approach ◽  
Evolutionarily Conserved ◽  
Import And Export ◽  
Transport Factors

Nuclear import and export is mediated by an evolutionarily conserved family of soluble transport factors, the karyopherins (referred to as importins and exportins). The yeast karyopherin Kap114p has previously been shown to import histones H2A and H2B, Nap1p, and a component of the preinitiation complex (PIC), TBP. Using a proteomic approach, we have identified several potentially new cargoes for Kap114p. These cargoes include another PIC component, the general transcription factor IIB or Sua7p, which interacted directly with Kap114p. Consistent with its role as a Sua7p import factor, deletion of KAP114 led to specific mislocalization of Sua7p to the cytoplasm. An interaction between Sua7p and TBP was also detected in cytosol, raising the possibility that both Sua7p and TBP can be coimported by Kap114p. We have also shown that Kap114p possesses multiple overlapping binding sites for its partners, Sua7p, Nap1p, and H2A and H2B, as well as RanGTP and nucleoporins. In addition, we have assembled an in vitro complex containing Sua7p, Nap1p, and histones H2A and H2B, suggesting that this Kap may import several proteins simultaneously. The import of more than one cargo at a time would increase the efficiency of each import cycle and may allow the regulation of coimported cargoes.

scholarly journals Immunoglobulin-binding domains: Protein L from Peptostreptococcus magnus

2003 ◽  
Vol 31 (3) ◽  
pp. 716-718 ◽  
Author(s):  
N.G. Housden ◽  
S. Harrison ◽  
S.E. Roberts ◽  
J.A. Beckingham ◽  
M. Graille ◽  
...  
Keyword(s):  
Binding Sites ◽  
Protein A ◽  
Cell Wall Protein ◽  
Protein G ◽  
Protein L ◽  
Heavy Chains ◽  
Binding Domains ◽  
Peptostreptococcus Magnus ◽  
Immunoglobulin Binding

Protein L is a multidomain cell-wall protein isolated from Peptostreptococcus magnus. It belongs to a group of proteins that contain repeated domains that are able to bind to Igs without stimulating an immune response, the most characterized of this group being Protein A (Staphylococcus aureus) and Protein G (Streptococcus). Both of these proteins bind predominantly to the interface of CH2-CH3 heavy chains, while Protein L binds exclusively to the VL domain of the κ-chain. The function of these proteins in vivo is not clear but it is thought that they enable the bacteria to evade the host's immune system. Two binding sites for κ-chain on a single Ig-binding domain from Protein L have recently been reported and we give evidence that one site has a 25–55-fold higher affinity for κ-chain than the second site.

scholarly journals The kinetoplastid kinetochore protein KKT4 is an unconventional microtubule tip–coupling protein

2018 ◽  
Vol 217 (11) ◽  
pp. 3886-3900 ◽  
Author(s):  
Aida Llauró ◽  
Hanako Hayashi ◽  
Megan E. Bailey ◽  
Alex Wilson ◽  
Patryk Ludzia ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Binding Activity ◽  
Significant Similarity ◽  
Load Bearing ◽  
Kinetochore Protein ◽  
Microtubule Binding ◽  
Binding Domains ◽  
Microtubule Binding Domain ◽  
Coupling Protein

Kinetochores are multiprotein machines that drive chromosome segregation by maintaining persistent, load-bearing linkages between chromosomes and dynamic microtubule tips. Kinetochores in commonly studied eukaryotes bind microtubules through widely conserved components like the Ndc80 complex. However, in evolutionarily divergent kinetoplastid species such as Trypanosoma brucei, which causes sleeping sickness, the kinetochores assemble from a unique set of proteins lacking homology to any known microtubule-binding domains. Here, we show that the T. brucei kinetochore protein KKT4 binds directly to microtubules and maintains load-bearing attachments to both growing and shortening microtubule tips. The protein localizes both to kinetochores and to spindle microtubules in vivo, and its depletion causes defects in chromosome segregation. We define a microtubule-binding domain within KKT4 and identify several charged residues important for its microtubule-binding activity. Thus, despite its lack of significant similarity to other known microtubule-binding proteins, KKT4 has key functions required for driving chromosome segregation. We propose that it represents a primary element of the kinetochore–microtubule interface in kinetoplastids.

The red cell band 3 protein: its role in anion transport

1982 ◽  
Vol 299 (1097) ◽  
pp. 497-507 ◽  
Keyword(s):  
Binding Sites ◽  
Band 3 ◽  
Anion Transport ◽  
Anion Binding ◽  
Hydrophobic Residues ◽  
Ping Pong ◽  
Hydrophilic Residues ◽  
Aqueous Core ◽  
Positively Charged

Studies of anion transport across the red blood cell membrane fall generally into two categories: (1) those concerned with the operational characterization of the transport system, largely by kinetic analysis and inhibitor studies; and (2) those concerned with the structure of band 3, a transmembrane peptide identified as the transport protein. The kinetics are consistent with a ping-pong model in which positively charged anion-binding sites can alternate between exposure to the inside and outside compartments but can only shift one position to the other when occupied by an anion. The structural studies on band 3 indicate that only 60 % of the peptide is essential for transport. That particular portion is in the form of a dimer consisting of an assembly of membrane-crossing strands (each monomer appears to cross at least five times). The assembly presents its hydrophobic residues toward the interior of the bilayer, but its hydrophilic residues provide an aqueous core. The transport involves a small conformational change in which an anion-binding site (involving positively charged residues) can alternate between positions that are topologically in and topologically out.

scholarly journals The Tail of XCTK2 Contains Two Distinct Microtubule Binding Domains

2020 ◽  
Vol 118 (3) ◽  
pp. 430a
Author(s):  
Stephanie C. Ems-McClung ◽  
Stephanie Zhang ◽  
Mackenzie Emch ◽  
Claire E. Walczak
Keyword(s):  
Microtubule Binding ◽  
Binding Domains
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