Driving Force for Enzymology

Enzymologists are beginning to turn their attention to energy-dependent protein machines. These multiprotein enzyme complexes drive a myriad of biological processes, including protein degradation, muscle contraction, and the delivery of proteins to their appropriate cellular homes.

Enzymologists have traditionally left the study of these macromolecular machines to biologists, in part due to their complexity. In a series of talks at the 19th biennial Enzyme Mechanisms Conference held earlier this month in Asilomar, Calif., enzymologists were reminded that these protein machines "are enzymes at heart," according to Irene Lee, an assistant professor of chemistry at Case Western Reserve University.

These biological machines are like car engines, Lee said. They consume a chemical fuel, usually adenosine triphosphate (ATP), and use the energy released to perform mechanical work, typically resulting in a biologically useful conformational change.

For a long time, scientists had assumed that the energy needed to drive such conformational changes came exclusively from the energy released upon hydrolysis of ATP's high-energy -phosphodiester bond. More recent evidence suggests that the simple binding energy of ATP may also play a role. Sorting out how ATP is used to perform mechanical work and exactly how much ATP is needed to do the job remains a challenge to those studying energy-dependent protein machines.

"Enzymologists are well prepared to ask these kinds of detailed mechanistic questions," said Lee, whose own lab studies an ATP-powered protein machine thought to selectively degrade oxidatively damaged proteins in humans. Lee chaired a session on protein machines at the enzyme mechanisms meeting in Asilomar.

In that session, Massachusetts Institute of Technology biology professor Robert T. Sauer described his lab's attempts to understand the mechanism of a simple bacterial relative of the eukaryotic proteasome, an ATP-powered machine charged with disposing of unwanted cellular proteins. This model bacterial proteasome (ClpXP) consists of a ring-shaped ATP-powered enzyme (ClpX) that unfolds unwanted proteins and feeds them into a barrel-shaped protease enzyme (ClpP), where they are chopped into small peptides.

Sauer's lab had shown that ClpX picks its protein targets by recognizing the specific terminal peptide tag that marks unwanted proteins for destruction. "The tag allows this machine to know what to degrade and what to leave alone," Sauer said. ClpX then hydrolyzes its bound ATP to drive a conformational change that begins to pull the tag--and the attached protein--into the pore. This mechanical force denatures the protein, and further rounds of ATP hydrolysis power the translocation of the unfolded peptide chain into ClpP's inner pore for degradation.

So just how much ATP is required to degrade a protein? To answer this question, Sauer and long-term collaborator Tania A. Baker of MIT have studied ClpX's ability to unfold and translocate titin, a small protein whose susceptibility to mechanical unfolding has been well characterized [Cell, 114, 511 (2003)]. By comparing the overall ATP consumption during degradation of titin and less stable titin mutants, they were able to estimate how much ATP is used to unfold this particularly stable protein and how much is used to translocate it: more than 500 ATPs and about 100 ATPs, respectively. Although ATP requirements will differ for different proteins, these experiments show that "protein degradation by ClpXP can be very costly, often using as much or even more energy than was expended to make the protein in the first place," Sauer concluded.

Another speaker, Stuart S. Licht, described his lab's progress toward explaining how ClpP digests the proteins it's fed. Licht, an assistant professor of chemistry at MIT, is trying to measure the sizes of the peptide fragments that this protease spits out. He reported that his preliminary data suggest that, unlike trypsin or related proteases, ClpP does not simply cleave substrates at specific amino acid residues. Instead, it seems to start at one end and cleave substrates at every six to eight residues as they enter.

Other protein machines use ATP as fuel to ferry cargo around the cell. Zeynep Ökten, a graduate student in the lab of biophysicist James A. Spudich of Stanford University School of Medicine, described her efforts to track the movement of one such machine. Myosin VI is a wishbone-shaped enzyme that harnesses the energy of ATP to "walk" along filaments of actin, a biological polymer that crisscrosses cells and acts as a sort of intracellular highway.

Myosin VI is thought to be responsible for transporting a variety of different kinds of cargo within cells. It has remained unclear exactly how this enzyme moves along the actin filament. Most types of myosins walk hand-over-hand, where the enzyme's two "hands" take turns in the lead. Certain peculiarities in the structure of myosin VI, however, have led some scientists to suggest that it might move like an inchworm, where one hand always leads the way.

To clear up this issue, Ökten and Spudich attached a fluorescent label to one hand of the wishbone. They then used this dye to track the movement of a single hand of the wishbone when the enzyme is fed ATP. The step size they observed--72 nm--indicates that myosin VI also walks hand-over-hand along actin, Ökten reported [Nat. Struct. Mol. Biol., 11, 884 (2004)]. Just as for the ClpXP model proteasome, the precise molecular details of how myosin VI utilizes the energy of ATP hydrolysis to power protein movement remain to be worked out.

ATP may be the most widely used fuel for protein machines, but it's not the only one. Many of the cell's protein machines run on guanosine triphosphate (GTP) instead of ATP. At the meeting, Peter Walter, a Howard Hughes Medical Institute investigator and professor of biochemistry and biophysics at the University of California, San Francisco, described a GTP-driven machine involved in targeting proteins to the endoplasmic reticulum. Because nearly all proteins are synthesized in the cytoplasm, cells have worked out mechanisms to deliver proteins to their appropriate subcellular compartment. In eukaryotes, a GTP-bound ribonucleoprotein complex known as the signal recognition particle (SRP) binds to a signal sequence of a nascent, ribosome-bound polypeptide. SRP then proceeds to bind to its GTP-bound receptor on the outer membrane of the endoplasmic reticulum, dragging its ribosomal cargo along with it. Once bound to its receptor, SRP passes the nascent, ribosome-bound polypeptide to a nearby protein machine that shuttles the polypeptide into the endoplasmic reticulum.

Hoping to sort out exactly how SRP and its receptor use each GTP to power protein targeting to the endoplasmic reticulum, Walter and postdoc Shu-ou Shan have focused their attentions on Ffh and FtsY, simpler bacterial versions of SRP and its receptor, respectively. In collaboration with UCSF crystallographers Robert M. Stroud and Pascal F. Egea, they solved a 1.9-Å crystal structure of Ffh bound to FtsY, in which both enzymes contained nonhydrolyzable analogs of GTP [Nature, 427, 215 (2004)]. The structure shows that the two enzymes form a composite active site at their interface, where each enzyme can reciprocally activate the hydrolysis of its partner's GTP molecule. This surprising mechanism involves direct hydrogen bonding between the two bound GTPs and is thus completely different from that proposed for other GTP-powered machines, Walter pointed out.

ON THE BASIS of enzymatic analyses of mutant versions of Ffh and FtsY, Walter suggested that the targeting complex undergoes a series of conformational rearrangements before GTP hydrolysis triggers release of its protein cargo [PLoS Biol., 2, e320 (2004)]. Each of these rearrangements could afford the cell an opportunity to double-check that the right protein is being delivered to the right place before GTP hydrolysis breaks up the targeting complex, he proposed.

MIT chemistry professor and meeting organizer JoAnne Stubbe admitted that her goal in inviting these particular speakers was to urge enzymologists to begin to think about protein machines, systems that have largely been left to biologists to sort out. "There's still a lot we don't understand about these systems," she said. Particular areas where enzymologists might be able to lend their expertise include measuring ATP requirements and teasing out whether the binding energy or hydrolysis energy of a nucleotide triphosphate drives a given conformational change and the kinetics of the process, she said. "It's time to understand these systems in a more quantitative way."

THE BIG PICTURE
Setting The Stage For Probing Mechanisms Of Multiprotein Enzyme Complexes

Enzymologists have traditionally devoted themselves to understanding the detailed chemical mechanisms of single-protein enzymes. Most key cellular processes, however, are carried out by far more elaborate multiprotein enzyme complexes, and enzymologists are beginning to take note. At the 19th biennial Enzyme Mechanisms Conference in Asilomar, Calif., that shift was apparent.

"The focus of enzymologists has been turned away from individual proteins and onto proteins that function in ensembles to carry out the inner workings of cells," conference organizer JoAnne Stubbe, an enzymologist at Massachusetts Institute of Technology, pointed out. Such complex multiprotein assemblies are responsible for protein synthesis and degradation, DNA synthesis and repair, natural product biosynthesis, and a host of other cellular processes.

The availability of structures of all or part of these multiprotein enzymes has opened the door for enzymologists to probe their mechanisms, but the task won't be easy. Tracking the fate of a multiprotein enzyme's substrates and products--which are typically proteins--is far more challenging than following small-molecule substrates and products of individual enzymes.

"Many examples of inroads into understanding these complex systems were provided at the meeting," Stubbe told C&EN. In particular, she said, enzymologists are striving to develop new tools to probe the enzymology of these complex machines.

At the meeting, Neil L. Kelleher, an assistant professor of chemistry at the University of Illinois, Urbana-Champaign, showed that high-field mass spectrometry can be used to measure the rates of formation and disappearance of covalently bound intermediates in multiprotein enzymes that carry out the biosynthesis of certain natural products. Chemistry professor Stephen J. Benkovic of Pennsylvania State University chronicled his lab's efforts to use single-molecule fluorescence spectroscopy to study the assembly of a relatively simple viral relative of the DNA replisome, the multiprotein enzyme that catalyzes DNA replication in humans. These and other structural and biophysical tools should pave the way for studying the mechanisms of other multiprotein enzyme complexes.