Home | People| Research |Gallery | Publications | Equipment | Teaching| Work with us | Contact |Links


Research

Molecular Motors

Most biological processes require energy. The source of energy can be the light (in photosynthetic organisms), an electrocehmical gradient (eg., ATP synthase) or a chemical gradient (eg., ATP hydrolysis). Cells are equiped with a variety of nanomachines which are able to couple the energy released by ATP hydrolysis with a mechanical work. These nanomachines contain linear (miosin, kinesin, dinein) or rotary motors (helicases, bacteria flagellum). The best characterized motor is the ATP synthase. Direct observation of this motor allow the characterization of the physical parameters of this machine, uncovering amazing properties, far beyond any human-made device. Hence, the study of these biological motors is essential for the engineering of devices with applications in nanobiotechnology and nanomedicine. (Press here for more information on ATP synthase).

Unwinding the mechanism of DNA and protein translocases

RecA/AAA+ proteins are motor ATPases Associated to a large variety of cellular Activities (i.e. DNA unwinding, protein folding, DNA transport…). Interestingly, all members of this family share a striking structural similarity, which suggests they operate under a common mechanism and share a common evolutionary origin. Understanding how these hexameric motors emerged during evolution is a difficult task. It has been argued that the F-type ATPases emerged as a modular enzyme that was formed by the combination of a RNA or DNA helicase and a proton channel. An evolutionary scenario has been proposed in which their ancestors were membrane translocases that, initially coupled ATP hydrolysis to RNA/DNA translocation across the membrane and, subsequently, to protein translocation (Cabezon et al., 2012)(see figure 1). The evolution of an F-ATPase from the hypothetical protein translocase could have resulted from mutations that increased the hydrophobicity of the inner space of the ring, impeding protein translocation. With the transported protein trapped in the translocase, the torque generated by ATP hydrolysis would have caused the rotation of the entire central oligomer relative to the membrane channel (Mulkidjanian et al., 2007).    
   


Figure 1. Proposed evolution of membrane motor ATPases. Evolution of membrane-anchored DNA and protein translocases. Comparison of single-stranded DNA pumps, such as TrwB-like coupling proteins (a), double stranded DNA transporters of the FtsK/SpoIIIE protein family (b), protein/effector (purple) translocators like VirB4-like proteins(c) and F-type ATPases (d).

   

Molecular motors working on G-quadruplex DNA structures
Nucleic acid sequences which are rich in guanines are capable of forming four-stranded structures called G-quadruplexes (also known as G4-DNA). These consist of a square arrangement of guanines (a tetrad), stabilized by Hoogsteen hydrogen bonding and monovalent cations in the center of the tetrads. They can be formed of DNA or RNA, and may be intra- or intermolecular. Telomeric sequences consist of many repeats of the sequence d(GGTTAG), and the quadruplexes formed by this structure have been shown to decrease the activity of the enzyme telomerase, which is responsible for maintaining the length of telomeres. This is an active field for drug discovery, due to its implications in cancer and cellular aging. Recently, there is also an increasing interest in quadruplex in locations other than the telomere. G-quadruplex structural motifs are widespread in genomes and might be control elements in transcription, DNA replication and other areas of DNA metabolism (2). These structures are induced and stabilized by proteins. A number of naturally occurring proteins have been identified which selectively bind to G-quadruplexes. On the other hand, these structures also have to be dissociated by specific helicases to allow a correct replication and transcription process. An example of this group of helicases is the RecQ helicase family.


Figure 2. Structure of the intramolecular human telomeric G-quadruplex in potassium solution (PDB ID 2HY9)

   

TrwB is a DNA-dependent ATPase involved in DNA transport during bacterial conjugation (Tato et al., 2005). The structure of the cytoplasmic domain of TrwB, reveals a hexamer with a 6-fold symmetry and a central channel of about 20 Å in diameter (Gomis-Ruth et al., 2001). The protein presents structural similarity to hexameric molecular motors such as F1-ATPase, FtsK or ring helicases, suggesting that TrwB also operates as a motor, using energy released from ATP hydrolysis to pump ssDNA through its central channel (Cabezon & de la Cruz, 2006). Recently, we have shown that TrwB protein is a structure-specific DNA binding protein with very high affinity for G4 DNA structures. We have found that TrwB binds G4 DNA with 100-fold higher affinity than ssDNA of the same sequence. Moreover, the protein is at least 25 times more efficient in hydrolyzing ATP in the presence of this type of substrate and it forms hexameric complexes only with G-quadruplex DNA structures (Matilla et al., 2010). TrwB might be involved in resolving G4 secondary structures that arise during conjugative DNA processing, disrupting such structures to allow ssDNA transport through the central channel of the protein. In summary, we believe that TrwB is an exceptional model to study the mechanism of this type of motors on structured DNA, since the protein shares with other related molecular motors the capability to bind specific DNA structures that are important to its physiological roles.

From DNA transporters to protein transporters: further insights into an evolutionary scenario. Type IV secretion systems (T4SS) mediate the transfer of DNA and protein substrates to target cells. As mentioned above, conjugative systems codify for both, DNA and protein translocases, named TrwB and TrwK, respectively, in plasmid R388. All T4SS contain a TrwK-like protein, whereas TrwB is only conserved in T4SS that transfer DNA. Recently, the atomic structure of the C-terminal domain of a TrwK-homologue has been solved (Wallden et al., 2012). revealing a striking structural similarity with TrwB. Moreover, in our group, we have obtained the structure of the full length protein by single-particle electron microscopy, consisting of a hexameric double ring with a barrel-shaped structure (Peña et al., 2012). These findings suggest that TrwK, as TrwB, works also as a RecA/AAA+ ATPase. We have characterized the ATPase activity of TrwK (Arechaga et al. 2008). TrwK presents three alpha -helices in the C-terminus that are conserved in all TrwK-homologues but absent in TrwB. We have demonstrated that those helices play an important regulatory role, inhibiting the ATPase activity of the enzyme (Peña et al., 2011).Moreover, this autoinhibitory regulation mechanism seems to be a general feature in a large variety of ATPases to prevent futile energy waste and, interestingly, this inhibitory mechanism resembles that of F1-ATPase by its alpha-helical inhibitory protein IF1 (Cabezon et al., 2003).Binding and hydrolysis of ATP give rise to conformational changes that, in the case of TrwB and TrwK, would be associated to DNA or protein transport across the membrane, respectively. In spite of not sharing a significant homology in their amino acid sequences, the similarity in their structure indicate a common evolutionary origin and, therefore, they constitute an exceptional model to study the specific domains involved in each type of transport: DNA or protein. Interestingly, we have shown that like TrwB, TrwK is able to bind DNA with a higher affinity for G-quadruplex structures than for single-stranded DNA (Peña et al., 2012).


Molecular Motors Group - Elena Cabezón and Ignacio Arechaga
. Instituto de Biomedicina y Biotecnología de Cantabria. Albert Einstein 21, PCTAN 39011 Santander (Spain). Tel. +34 942 202033