SNARE Crystal Structure

For this article, Eugene Russo interviewed Axel T. Brunger, a Howard Hughes Medical Institute investigator and professor of molecular and cellular physiology at Stanford University. Data from the Web of Science (ISI, Philadelphia) show that Hot Papers are cited 50 to 100 times more often than the average paper of the same type and age. R.B. Sutton, D. Fasshauer, R. Jahn, A.T. Brunger, "Crystal structure of a SNARE complex involved in synaptic exocytosis at 2.4 angstrom resolution," Nature, 395:

Nov 27, 2000
Eugene Russo

For this article, Eugene Russo interviewed Axel T. Brunger, a Howard Hughes Medical Institute investigator and professor of molecular and cellular physiology at Stanford University. Data from the Web of Science (ISI, Philadelphia) show that Hot Papers are cited 50 to 100 times more often than the average paper of the same type and age.

R.B. Sutton, D. Fasshauer, R. Jahn, A.T. Brunger, "Crystal structure of a SNARE complex involved in synaptic exocytosis at 2.4 angstrom resolution," Nature, 395: 347-53, Sept. 24, 1998. (Cited in about 210 papers since publication)

The precise role of SNAREs, protein complexes known to be integral to cell membrane fusion--and, as a result, a number of cellular functions1--continues to elude researchers. By revealing the complex's X-ray crystal structure, however, this paper has helped scientists devise appropriate SNARE experiments that continue to elucidate the protein.

SNAREs (soluble NSF-attachment protein receptors; NSF stands for N-ethylmaleimide-sensitive fusion protein), which are evolutionarily conserved in organisms from yeast to man, mediate the fusion of transmitter-carrying vesicles with their target membranes. SNAREs, for example, help shuttle neurotransmitter-filled vesicles to synapses, an important step in the ensuing communication between neurons. SNAREs form a complex that fuses the vesicles to the cell membrane, thus triggering neurotransmitter release. A group led by senior author Axel T. Brunger, then at Yale University, used X-ray crystallography to produce the first high-resolution images of SNAREs as they propelled neurotransmitters toward their destination. Choosing rat SNAREs as their model, investigators revealed a highly grooved synaptic fusion complex surface with distinct hydrophilic, hydrophobic, and charged regions thought to be important for membrane fusion. Prior images had used only low-level electron microscopy.

"With the structure, [researchers] were able to put things on a more solid ground in terms of designing experiments and making models," remarks Brunger, now a Howard Hughes Medical Institute investigator and professor of molecular and cellular physiology at Stanford University. Experimenters got a good idea of the protein's interaction sites. The structure, says Brunger, has had an impact on those scientists working on synaptic neurotransmission and on those working more generally on vesicle trafficking in systems such as yeast.

In collaboration with Reinhard Jahn, then at Yale, Brunger spent two years working out the biochemistry of the system--getting the proteins at the right constructs to express well, working out purification protocols, and trying to understand the folding tendencies and stability of the proteins. "Once we understood the system better, we actually got the crystals fairly quickly," says Brunger. Eight months after getting the crystals, they solved the actual structure. "These were difficult crystals, so it took quite some time to get them diffracting to high-enough resolution," Brunger explains. The stubbornness of the crystals has made it difficult to improve on the structure's 2.4 Angstrom resolution in the two years since this paper. According to Brunger, the flexibility of the SNARE complex, evidenced by the crystal structure itself, provides an intrinsic limitation on the system's potential resolution.

The degree to which SNAREs directly participate in vesicle fusion is still a matter of debate; the complex could have a large role or could be preparing the system for fusion by something else. "How it works in molecular detail hasn't been fully worked out yet," comments Brunger. He notes that although finding the crystal structure was an important step, it only indicates the structure at some point after fusion has happened. Because the crystals grow over an extended time period, researchers cannot capture transient assembly states of the SNARE complex, some of which may be key to the process of membrane fusion. Nevertheless, since the structure came out, says Brunger, researchers have "started, a lot more intensively than before, to carry out experiments on these proteins." A group headed by James E. Rothman of the Memorial Sloan-Kettering Cancer Center in New York, for example, has demonstrated in vitro fusion with only SNAREs.1,2

Brunger's group is focused on acquiring the crystal structures of SNARE accessory proteins such as synaptotagmin, vesicle fusion proteins called Rab proteins, and NSF enzymes, chaperones that disassemble the SNARE complex. Of particular interest: finding the structure of super complexes between these proteins and the SNARE complex.

 

Eugene Russo can be contacted at erusso@the-scientist.com.

References

1. J. Kling, "Explaining Membrane Fusion," The Scientist, 14[6]:18, March 20, 2000.

2. T. Weber et al., "SNAREpins are functionally resistant to disruption by NSF and alpha SNAP," Journal of Cell Biology, 149:1063-72, May 29, 2000.