Beset by peers trying to tear them apart, proteins known as proteases constantly risk destruction. UCSF scientists have determined how a nearly impregnable design protects some of the most besieged proteases, a design that contradicts a basic assumption of chemistry.
The UCSF team studied alpha-lytic protease, a protein that breaks down nutrients for bacteria in the soil, an unforgiving environment where hundreds of protease species slash at each other. They found that the alpha-lytic protease is about 100 times more resistant to attack by other proteases than are digestive enzymes or other conventional proteases.
The key to this toughness, the scientists discovered, is the novel way the protease is folded into working order, a process fundamentally different from the effortless way most proteins fold into action – and one that requires more energy at the outset but achieves remarkable stability.
They suspect this design strategy is mimicked in other environments extremely vulnerable to protease destruction, including lysosomes in human cells, where proteins are broken down for recycling.
The UCSF research is published in the January 17 issue of the journal Nature.
Most proteins — made of linear chains of amino acids – fold spontaneously into their working configuration, a shape that represents their most relaxed condition — their lowest “energy state.” They occasionally unfold to some degree and then refold — "breathing" as chemists call it. Whenever they do unfold, proteins risk attack by proteases designed to tear at newly exposed regions.
But the alpha-lytic protease does not spontaneously fold into a ready-to-work shape. It must be bent into working condition by a catalyst that temporarily becomes part of the molecule and is known as the pro-region. The UCSF researchers found that the structure sculpted by the pro-region is remarkably resistant to unfolding. Whereas normal proteins transiently unfold in minutes to days, alpha-lytic protease can take a year or more to unfold, making it extraordinarily resistant to proteolysis, or breakdown by other proteases.
The slow unfolding, the scientists found, is the result of an unprecedented rigidity in the folded state, a rigidity that comes at an energetic cost. In physics terms, the protein is not stabilized as most proteins are by thermodynamics – the natural relaxation into its lowest energy state – but by kinetics, or being physically trapped in a higher energy state by the action of the pro-region. When the folding is complete, the pro-region is degraded, and the alpha-lytic protease remains locked in its high-energy functional form.
"This novel way of achieving protein folding is apparently nature’s ultimate strategy to protect proteins from being digested by proteases," said David Agard, PhD, senior author on the paper and a Howard Hughes Medical Investigator at UCSF. Agard is UCSF professor of biochemistry and biophysics.
To measure how well defended alpha-lytic protease is to protease attack, the researchers used the technique of magnetic resonance spectroscopy (NMR) to test the ability of water to penetrate the protease molecule. They found the degree of protection is not only among the highest ever measured but that the most defended regions of the molecule are spread throughout the protein, providing a rigidity to its shape and resistance to unfolding "well beyond that seen for traditional thermodynamically stabilized proteins."
“These studies have allowed us to look at protein folding not just as the means to the folded state, but as a process that determines how proteins function,” Agard said.
“The most remarkable aspect of this system is that the evolution of the pro-region has allowed the folding pathway, requiring the pro-region, to be completely divorced from the unfolding pathway. This in turn has removed ‘classic’ stability requirements on the folded protein, permitting new functions to evolve.”
The unusual folding process allows the protein to exist first under one set of constraints — one "energetic landscape," as the scientists call it — and then another, changing both its shape and its working opportunities in the process. The researchers suspect this novel folding route probably appears often where proteins are extremely vulnerable to proteolytic attack. Many other proteases contain the distinctive pro-region, the scientists note, including all of the secreted bacterial proteases and the recycling proteases found in lysosomes of higher cells, “strongly suggesting” that these other proteases are folded by the same process that shapes the alpha-lytic proteases, Agard said.
University of California – San Francisco. January 2002.