Let’s get acquainted with another irreducibly complex molecular motor. This one is a master at unfolding proteins, even the tough nuts that are hard to crack. Its method is ingenious.

One shouldn’t think that icons of intelligent design in biochemistry are restricted to the few favorites, like the bacterial flagellum, kinesin, and ATP synthase. There are thousands of them — tens of thousands! Each one is fascinating in its own way. Today’s focus is on one, called the “Cdc48 ATPase complex,” or Cdc48 for short. It’s another ATPase (meaning, it uses ATP for energy) with an important job: unfolding proteins. Most proteins, you recall, are folded into globular shapes. Some of those can get pretty tightly wound, held together with electrical charges and mechanical forces. Anyone who has tried to untangle threads, ropes, or fish lines knows that the tighter the wad, the harder it is to untangle. Here is how Cdc48 does its job — with finesse.

Ubiquitin, as in Ubiquitous

Before getting into the mechanics of Cdc48, it would help to learn about another protein called ubiquitin. As the name implies, it is ubiquitous in the cell. Because ubiquitin fastens easily to many domains on other proteins, it often “tags” them so that they get noticed by other enzymes. Ubiquitins can be chained together easily, conferring additional tagging messages. Some poly-ubiquitin tags tell the proteasome (the cell’s garbage disposal) that the substrate needs to be recycled. The proteasome removes the poly-ubiquitin tag before stuffing the spent or damaged protein into the barrel-shaped slicer-dicer, where its amino acids are separated for recycling.

Another poly-ubiquitin tag informs Cdc48 that “this substrate needs unfolding.” Such a situation can occur when a protein is too tightly wound for the proteasome, when it is part of a multi-protein complex, or when it is in a hard-to-access area, such as embedded in a membrane. What happens when the protein fastens to the enzyme is quite amazing. The action was recently deciphered in more detail than ever by Edward Twomey et al. who describe their findings in Science. Get ready for a trip down a spiral staircase inside another molecular machine! 

Visualize Its Shape

To visualize its shape, think of two hexagonal tires stacked on top of each other. A hand from inside grabs the end of the first ubiquitin, and pulls on it. Out the bottom tire comes an unfolded protein. Presto! What happened inside?

Cdc48/p97 belongs to the AAA+ (ATPases associated with diverse cellular activities) family. It contains an N-terminal domain and two tandem ATPase domains (D1 and D2). Six monomers form a double-ring structure, with a central pore. Cdc48 often collaborates with the heterodimeric cofactor Ufd1/Npl4. Substrate initially binds through the attached polyubiquitin chain to Ufd1/Npl4 and then moves through the pore of the ATPase rings and is thereby unfolded. This translocation process requires adenosine 5′-triphosphate (ATP) hydrolysis by the D2 domains and involves their pore loop residues. [Emphasis added.]

In short, Cdc48 literally pulls its substrate through a central pore with force. The pore, they say, is shaped like a spiral staircase. Finely-placed amino acids that look like loops within the pore grab onto the substrate and pull it through in a “hand-over-hand” manner. It’s a powered string untangler!

Where does the ubiquitin come in? The machine grabs the poly-ubiquitin “tag” first, and pulls it into a special groove in the machine. This gives Cdc48 both a signal to proceed, and also something to grab onto for recognizing the substrate coming in behind. 

An Amazing Motor

Twomey et al. wanted to know more about this amazing motor.

The mechanism of substrate processing by Cdc48 is poorly understood. For example, it is unknown how Cdc48 can deal with a broad range of even well-folded substrates, the only requirement being an attached polyubiquitin chain. Specifically, it remains unclear how a segment of a folded substrate can pass through the D1 ring to contact the D2 pore loops that power translocation. How these D2 subunits then translocate the substrate is also not known. Structures of related hexameric ATPases indicate a spiral-staircase arrangement of pore loops around the substrate, but structures of the Cdc48 ATPase engaged with a polyubiquitinated substrate have not yet been determined.

The biochemists found a way to slow down the action so that they could watch it. To do this, they had to mutate the enzyme so that it didn’t operate so fast. Then, they captured “movie frames” using cryo-electron microscopy. Then they could put the frames together and see what was going on.

The structures show two folded ubiquitin molecules bound to Npl4 located on top of Cdc48’s D1 ring. Hydrogen-deuterium exchange experiments indicate additional ubiquitin-binding sites on Ufd1. Surprisingly, one ubiquitin molecule is unfolded and bound to a groove of Npl4, which contains conserved amino acids required for substrate unfolding. Unfolding of ubiquitin is remarkable, given that it is an extremely stable protein that can survive boiling. The unfolded ubiquitin molecule projects its N-terminal segment through the D1 ATPase ring and engages the pore loops of the D2 ATPases. These pore loops form a staircase that acts as a “conveyer belt” to move the polypeptide through the central pore.

Illustrations in the paper show the ubiquitins fitting tightly into a special groove made to order for them. The machine unfolds the first ubiquitin (like they say, quite a feat for a stable protein that can survive boiling!). This gives the substrate that follows a “handle” that the machine can pull on. The ubiquitins engage both the specialized groove in D1, but also the “conveyor belt” in D2, where a spiral staircase of six loops named A to F pull on the substrate, unfolding it in a hand-over-hand manner. The sequence of peptides coming in doesn’t matter to this general-purpose unfolding machine; it can handle them all. Particularly tough nuts may require additional ubiquitin “leader” chains:

Our results explain why the Cdc48 ATPase can act on a broad range of even well-folded proteins: It uses ubiquitin binding and unfolding to initiate substrate processing. Cdc48 first pulls on the N terminus of the unfolded ubiquitin molecule. The structure implies that if substrate is directly attached to the unfolded ubiquitin, it will next translocate through the central pore; otherwise, Cdc48 has to successively unfold the intervening ubiquitin molecules until it reaches and unfolds the substrate.

Why the Machine Is Important

This machine is important. Defects in Cdc48 can cause neurological diseases. Surely this must be a crown product of late evolution, right? No; the scientists did their work with yeast, one of the very simplest eukaryotes. And it is also found in archaea, considered by some to be the most primitive life forms on earth. One could rightly suspect that without this general-purpose untangler present at the beginning, the first cells would quickly become clogged with tangled wads of useless polypeptides. 

Need more amazement? The scientists found that this machine has moving parts. If you recall those old hand-operated label-makers, it’s a bit like that: the user would crank a handle to send the tape moving along with each letter. Here’s what the researchers found:

The D1 and D2 domains both behave as rigid bodies (fig. S11). Superposition of the Cdc48 monomers on the basis of the D1 domains shows that the staircase arrangement of the D2 domains is caused by rigid body movements relative to D1 (fig. S11B). The angle between the D1 and D2 subunits changes dramatically from subunits B to E, causing a displacement of the D2 pore loop by more than 18 Å (fig. S11B).

That’s a pretty large motion for a molecule. Here is how the general-purpose unfolding machine handles any chain:

Five of the subunits (A to E) contact the polypeptide and form a pronounced staircase, with subunit A on top and subunit E on the bottom…. All substrate-engaged subunits contact the polypeptide through the Trp561 and Tyr562 residues in their pore loops (Fig. 6B). These residues “pinch” every other peptide bond of the extended polypeptide substrate. Thus, each power stroke of the hexameric ATPase moves two amino acids of the substrate, and the intercalation of pore loops between side chains allows translocation of a polypeptide regardless of its specific amino acid sequence.

Where to Grab

In other words, regardless of the shapes or sizes of the amino acids, the machine knows where to grab. It “feels” for the peptide bond common to all amino acid residues, pinches it, and pulls with a “power stroke,” resulting in a large motion between D1 and D2. The subunit loops are in contact with the substrate all the way through, making sure it unfolds properly. But why doesn’t the pulling motion dislocate the machine as well? Therein likes another wonder. Think of a conveyor belt. The belt eventually moves back to its starting position. 

Together, our structures reveal that the D2 ATPases use a “conveyor belt” mechanism to translocate a polypeptide chain through the central pore (Fig. 6D). In this model, subunit A binds to the top of the polypeptide chain when it converts from the ADP-bound or nucleotide-free state to the ATP-bound state. The subunits then move downward, dragging the polypeptide chain with them. At the lowest substrate-engaged position, subunit E hydrolyzes ATP. Subsequently, in the ADP-bound or nucleotide-free state, subunit F disengages from the substrate so that it can move to the top position and start a new cycle. 

This is so like macro-machines we are familiar with: conveyor belts, windmills, sewing machines, and other devices that continually reposition themselves for continuous action. Other molecular machines use a similar conveyor-belt mechanism, the authors say, but Cdc48 seems to be the deluxe model with an added contact that “may allow Cdc48 (p97) to exert more force to unfold its substrates.” This is not simple chemistry. This is machinery with moving parts, forces and interacting subunits!

As expected, the mechanism is even more complex than described here. And, again as expected, the authors have nothing to say about evolution.

Would They Dare?

Behe notes in his Appendix to Darwin Devolves that evolutionists have failed to explain the bacterial flagellum or the blood clotting cascade with a Darwinian mechanism. He gave them that challenge to Darwinism in Darwin’s Black Box twenty years ago. A couple of teams tried, but dodged the issue. The rest have remained silent. And yet they still keep their jobs, demanding sole authority for Darwinism in science. It’s time for some overkill: not just one or two molecular machines, but thousands of them, including Cdc48. Show them all, day after day, incessantly, faster and faster, until the most ardent Darwinist cries uncle. 

At first, if you have seen the old I Love Lucy episode, Lucy and Ethel could handle the conveyor belt of chocolates. They could excuse the occasional missed one, or swallow another. Let’s put Jerry Coyne and Richard Dawkins in their places and watch the fun as we send molecular machines down the conveyor belt for them to wrap in Darwinian explanations, and when they are really sweating, shout, “Speed ’er up, all!”

Photo credit: Jude Beck via Unsplash.