Computationalcience AllSfor

K computer Newsletter
March 2016

K computer Helps Solve Mystery of
Bacterial Drug Resistance
Explaining living phenomena
in physical terms

12No.

K computer Helps Solve Mystery of
Bacterial Drug Resistance
Explaining living phenomena in physical terms

Yasuhiro Matsunaga
Research Scientist with the Computational Biophysics Research Team, RIKEN AICS
AcrB as viewed from one side
Various drugs being discharged
from the cell
Outside the cell

WatWa
Watateerr momololecules
ecuecuules
leesesBindin
BiBindinngfoffororm

C: Simulation of the structural change(from the binding to discharging forms)

Extracellular membraneDrugDiﬀerent proteinsthat have penetrated
the extracellularmembrane

Various drug
molecules
Bindingform

Discharging
form
AcrB
Cellular membraneaneInside
Inside thethhe cellell

Diﬀerent proteinstranspportingg drugs
g

Cellular membrane

ProtonSite of proton binding

A: Roles of AcrB
neBacteria, including, have an extracellular membrane
heoutside the cellular membrane. AcrB, embedded in thendcellular membrane, ingests various drugs and toxins anddischarge them through diﬀerent proteins that have penetrated the extracellular membrane. This process is driven byhydrogen ions (H+: protons) ﬂowing into the cell throughthe lower part of AcrB.

DisschaDischhaargi
rg
ging
gingngform
fo

AcrB as viewed from above

Drug

Ingestinggform

Binding
dingform

The binding-todischarging form
causes the dischargeof drugs

Discharginggform

Binding
form

Discharging
gform

Ingesting
gform

Drug
B: Functional rotation
AcrB is a combination of three identical proteins. Each
protein undergoes a regular structural transformation̶theingesting, binding and discharging forms, in that order. Thisfunctional rotation process acts to force various drugs outof the cell smoothly. A good balance among the threeforms helps AcrBʼs overall stability.
Fig. 1 Multidrug eﬄux transporter AcrB protein

Pathogenic bacteria that have become resistant to medical drugs pose a serious
threat to clinical practice. In such cases, the drugs are driven out of the body by themultidrug eﬄux transporter AcrB protein embedded in the bacteriaʼs cellular
membrane and become almost ineﬀective.
To work out the mechanism causing the eﬄux or discharge of a drug, Matsunaga
simulated AcrBʼs functions on the K computer. He discovered that drug eﬄux takes
place as the protein undergoes a transformation when binding with just onehydrogen ion.

Infatuated withαWhen Matsunaga enrolled in theDepartment of Earth and Planetary
Sciences (EPS), Faculty of Science, Kobe

University, in 1997, he spent much of his
early campus life reading extensively.
During this period, he came across abook that would impact the rest of hiscareer. The title wasbyKeisuke Ito, who was a professor in EPS
at the time.
Itoʼs book triggered Matsunagaʼsinterest in Chaos Theory̶the phenomenon of how small changes in a system canlater lead to unexpected outcomes. “Ibecame drawn to computer simulationsof chaos because it was fun to see simulations produce unpredictable results one
after another,” he recalls. In obtaining hisMasterʼs degree working under Professor
Yukio Gunji and Associate Professor
Tamiki Komatsuzaki at EPS, he took up thestudy of simulation as a research tool in aserious way.
“Calculations using Newtonʼs equationof motion, (force) = (mass) xα(acceleration), makes possible a variety of simulations, from the orbits of planets to themovements of proteins,” says Matsunaga.
“Even in simple simulations, like those of
movements of planets in our solar-system, chaos could produce surprisingresults. As a young researcher, I became
infatuated with the charm of Newtonʼsα, and set myself the goal of mastering the secrets of the equation.”

AcrB responsible forbacterial resistanceto drugs
After earning a D. Sc. in information andmedia science, Matsunaga joined the labof Professor Akinori Kidera in Yokohama
City University in 2007 and was later
assigned to research AcrB using simulation.
AcrB is a protein embedded in bacterial
cellular membranes (Fig.1A). As moleculesof various drugs enter the cells of bacteria,
the protein ingests then discharges them,and in this way the bacteria develop aresistance to the drugs.
When Matsunaga began investigating
AcrB, previous researches had alreadyproduced a partial picture of the proteinʼsshape and the discharge mechanism.
Most proteins, including AcrB, have athree-dimensional structure composed ofa folded amino-acid chain. Never staticwithin a cell, a protein morphs into various structures to perform its functions.
These changes can be simulated using
calculations based on interatomic-forces
and theα equation. Vital to such simulations is information obtained at theatomic scale on the shape of the protein

Cellularmembrane

Deviated region in the positive direction
Without a proton

With a proton

Fig. 2 Change of electrostatic-potential (with and without one proton attached to AcrB in the binding form)
With one proton attached to AcrB in the binding form, the region deviated in the positive direction (shown in blue)
expands, resulting in loss of stability.

and on the kind and location of theatoms. The most common way to obtainsuch data is X-ray crystallography, thetechnique of applying X-rays to protein
crystals to analyze their structures.
Professor Satoshi Murakami, currently
at the Tokyo Institute of Technology, wasthe ﬁrst to reap major results from the
X-ray crystallography approach. In a 2002
analysis using RIKENʼs SPring-8 synchrotron radiation facility, he revealed AcrBʼsstructure and showed it to be a combination of three identical proteins.
In 2006, Professor Murakami followed
this revelation with a successful experiment, also conducted on the SPring-8,
that examined the structure of AcrB whena drug molecule is bound. He discoveredthat each of the three proteins mutuallydiﬀers slightly in structure, and that eachstructure corresponded respectively tothe forms that ingested, bound, anddischarged the drug molecules.
Professor Murakami surmised that each
AcrB protein changes its structure insequential order to perform these threeactions (Fig.1B). Then, what drives thissequential change?
It is known thatand other bacteria
have an extracellular membrane. And
between it and the cellular membrane
protons (hydrogen ions) are present inhigh concentrations. It is also known
that the protons ﬂow into bacterial cells
through the membrane proteins. Thus,
it seemed likely that AcrBʼs structuralchange takes place as the protons permeate through the proteinʼs lower
region immersed in the cellular membrane (Fig.1A).
In other research, Professor Mitsunori
Ikeguchi at Yokohama City University had
identiﬁed the binding site of protons thatwould mainly aﬀect AcrBʼs structure. It
remained to be seen, however, what theexact structural change was that caused

the discharge of drugs. Matsunaga wasgiven the task of answering this question
using computer simulation.
“AcrB is a complex organic compound,consisting of as many as 500,000 atoms
including those of the surrounding watermolecules and cellular membranes,”
Matsunaga explains. “Simulating its activity by evaluating interatomic forces andrepeating
α calculations entails anenormous amount of calculation.”
After joining AICS in 2011, Matsunaga
sought to devise eﬀective calculation
methods to simulate AcrB on the K computer. His eﬀorts were eventually rewarded. He succeeded in reproducing the process of structural change in AcrB when
the proteins bind with the protons̶theprocess that results in a discharge of drug
molecules (Fig.1C).

Can an ant annoy
an elephant?

AcrB proteins ingest and discharge
drugs in their upper region, which is relatively far from the lower region where theprotons permeate. “Itʼs hard to imagine
that a single, large ʻsystemʼ of 500,000
atoms would undergo a structuralchange̶from a binding structure to astructure that discharges foreign molecules̶merely upon the adhesion of justone proton,” says Matsunaga. “Figuratively speaking, itʼs like an elephant being
irritated by an ant walking over its foot.”
Matsunagaʼs simulation work produced
some surprising ﬁndings. When AcrB proteins in binding form are without protons,
practically no electrostatic-potential frustration appears in either the positive or
minus direction. Yet, when coupled withjust one proton, the proteins experienced
a major expansion of the region that devi-ates in the positive direction. This resulted in the proteins losing much of theirelectrostatic stability.
“A large stability loss from just one
attached proton!,” Matsunaga exclaims(Fig. 2) .
The simulations also showed AcrBʼs
lower region changing structurally, as if
to alleviate the frustration. This, in turn,aﬀected the upper region and resulted
in the structural change that discharged
the drug.
Previous research showed that somespeciﬁc amino acids in the proteinʼslower region also play a role in thisstructural change. “Given the amount ofstability loss, there must be otherimportant factors involved as well,” says
Matsunaga. “I am looking for these bycomparing the two states of AcrB in thebinding form: one with the single
proton, the other without.”

Seeking out
common features ofallostery
An inhibitor drug is any molecule thatbinds and deactivates a protein ultimately responsible for disease. As forAcrB, its drug-discharge functions may
be inhibited by deactivating not just thedrug-binding upper region but also thesite that informs the upper region of achange in the proton-attached lower
region.
Thus a better understanding of a proteinʼs functional mechanism should
help expand the number of options
when targeting sites with inhibitor
drugs. It is expected that the strategy oftargeting other sites than the obvious
binding sites will lead to the productionof new drugs for proteins previously
hard to deactivate.
The way AcrB functions represents aninteresting example of allostery.
Allostery refers to the process by whicha small change at one site causes achange in a separate site.
Thanks to allostery, a range of proteins are able to carry out their functions(Fig.3). “There should be a hidden
thread running through all this, and I
would like to determine what it is by

One surprising ﬁnding Matsunaga discovered during his research of AcrB was that it
took far more time to analyze the simulation data than to carry out the actual simulations using the K computer. This has led
him to seek out the help of machine learning and artiﬁcial intelligence to help speed
up this process.

way of simulation work,” says Matsunaga. “To this end, I'm looking to see howmachine-learning technology could
draw out common features in simulation data that we humans can miss.
These features will most likely assist
development of new drugs.”

New era of life science
openingCalculating the activity of proteins,
each consisting of a very large numberof atoms, requires an enormous amountof mathematical work and computer
processing time. As such, researchesʼ
ability to simulate protein activity has
been limited to mere microseconds(millionths of a second). Matsunaga,
however, using a new method of calculation together with the power of the K
computer has succeeded in extending
the amount of simulation time a thousand fold̶on the order of milliseconds(thousandths of a second).
“This now makes it possible to directlycompare the results of simulations andexperiments,” he says. “That will lead toa clearer understanding of life phenomena. Once we predict, through simulation, that a particular site in a protein
likely has a lot to do with the proteinʼsoverall functions, we should be able toverify the prediction by experimentsthat modify the site and examine thefunctional changes brought about by
those modiﬁcations.”
Though experimentation will enable
researchers to compare protein conditions before a structural change withconditions after the change, it is diﬃcult
to observe these changes in real time.
The same is true of AcrB.
“AcrB in the binding form, with one
proton attached, enters an unstablestate for an extremely brief period, toobrief for observation,” he points out.
“But, using simulation, we are able toexamine the key moments of proteinfunctions at the atomic scale andexplain life phenomena in physicalterms.”
Life science is now at the threshold of
another breakthrough, where computer
simulation serves as a primary tool ofresearch together with experiment.

ATP molecule andAMP moleculeFig. 3 An example of protein allostery
The enzyme adenylate kinase closes up as ATP and AMP molecules combine, causing a reaction. Due toallostery, the process by which a small change in one site causes changes in other sites, proteins often
become active.

RIKEN Advanced Institute for Computational Science (AICS)
7-1-26, Minatojima-minami-machi, Chuo-ku, Kobe, Hyogo, 650-0047, Japan
TEL: + 81 78 940 5555 FAX: + 81 78 304 4964 E-mail: aics-koho@riken.jp http://www.aics.riken.jp/en/