Tale

Right place, right time

Ernesto Fuentes

University of Iowa College of Medicine

Published January 29, 2021

As a kid, Ernesto Fuentes lived in Brazil just long enough to stock up on the soccer skills he now shares with his kids. His affinity for structural biology may have arisen at the same time, shaped by family trips to see churches and other architectural highlights in nearby countries. Little did he know at the time, he would grow up to bring fresh insights to common building blocks responsible for a dazzling diversity of normal molecular function in living cells.

Back in the United States, Fuentes attended college in Ohio. He intended to become a chemical engineer, like his dad. Then he learned that proteins, like buildings, have distinctive architecture. He pivoted to a biology masters degree and followed up with a PhD specializing in nuclear magnetic resonance (NMR).

His doctoral studies immersed him in NMR and dynamics in more ways than one. His mentor’s lab moved twice, from University of Illinois to State University of New York Buffalo and then to University of Pennsylvania.

As a postdoctoral fellow in a University of North Carolina cancer laboratory, Fuentes established a paradigm for his future lab: Collaborate with colleagues doing the biology.

That leaves Fuentes free to explore fundamental questions about how a common part of many proteins can be involved in so many specialized tasks inside cells.

His postdoctoral work introduced Fuentes to PDZ domains, a small protein region found in more than 250 proteins that cover a broad swath of biology. PDZ domains connect partner proteins to each other in large signaling structures inside a cell next to the membrane in an arrangement that facilitates specific biological action.

PDZ domains somehow put together the protein scaffolding “in the right place at the right time” to get things done, Fuentes says.

Thirty years ago, scientists figured out the basic function: A PDZ domain in one protein grabs another protein by its tail. The tail, or C-terminus, fits neatly into the main PDZ groove. But there are subtleties in that maneuver that dictate exactly what other proteins can connect and what they can do as a result, Fuentes and others are discovering as they develop new ways to learn the rules of the PDZ family specificity.

Since Fuentes joined the University of Iowa College of Medicine faculty in 2006, his lab has been probing the specialized interactions of PDZ domains.

In their research, the Fuentes lab applies multiple integrated structural biology techniques for deeper insight, including NMR, X-ray scattering, crystallography, and cryo-electron microscopy, leaning on a full repertoire of SBGrid software.

“We don’t do biology, but we’re really interested in biology,” Fuentes says. “We’re developing tools that allow us to interrogate biology. We can do that through structure.”

The PDZ domain is found in many proteins and a protein can have one or many PDZ domains. Not only is PDZ a popular domain in proteins, it is a veritable social butterfly. PDZ domains bind to their counterparts in other proteins and mix it up with other ligands. Learning how finely tuned PDZ domains meet up with binding partners will illuminate normal biological signaling and may help develop more precise therapeutics.

PDZ domains help build protein complexes that facilitate different types of cell adhesion and signaling. In particular, they allow epithelial and neuronal cells to form cell-cell adhesion complexes that are critical for normal tissue and neuronal function, Fuentes says. Mutations that affect PDZ activity can have health consequences, including cancer, deafness, neuropsychiatric conditions, and cystic fibrosis.

In a central question, the lab has been asking how selective are PDZ domains in their binding partners, also known as specificity.

To sum up a dozen years of work with three PDZ family members (CASK, TIAM1/2, and Scribble), Fuentes and his colleagues have found that the PDZ groove for a protein tail is just a starting point. Some of the specificity comes from dynamics in the other PDZ nooks and crannies known as subpockets. Not only can certain amino acids wiggle into place better than others on a PDZ domain, the PDZ subpockets will adjust themselves to accommodate a preferred protein partner.

A PDZ domain has multiple subpockets that can recognize and accommodate specific amino acids of target proteins. According to results of mutational experiments, PDZ subpockets show enhanced dynamics and cooperative energetics. Courtesy: E.Fuentes.

“Changes in the dynamics might be key to understanding specificity,” Fuentes says. For example, a protein with more dynamic range may be able to accommodate more partners and participate in different types of signaling. A less dynamic protein may have a more specific role. That means a mutation could allow more or less dynamic changes, influencing the biological system.

The researchers have experimented with mutations in different PDZ subpockets to observe the impact on binding. Some mutations sparked increased dynamics, also called hot spots, and those PDZ domains favored different or more binding partners.

“The really cool thing, this gave us insight into how evolution can change the specificity of a single PDZ domain,” Fuentes says. “Over time, mutations may be able to broaden or direct the domain to a new set of targets.”

In another twist, they found that some PDZ domains can bind internal motifs and not just the canonical C-terminal tail. “What’s interesting is that it opens up another set of interactions and set of proteins,” he says. “There are not that many examples, and the specificities are not well understood.”

Fuentes found a rare example in a collaboration with a cancer researcher who is studying how epithelial cells normally are held together so tightly. Most cancers originate in epithelial cells, and cancers have to overcome the cell-to-cell adhesion system to grow and spread.

In the project, Fuentes' team was tasked with dissecting an interaction between the proteins SGEF and Scribble involving a PDZ domain. They found an internal PDZ motif in SGEF that binds to one of Scribble’s PDZ domains.

In a collaborative project, a research team identified a new cell polarity complex composed of Discs large (Dlg), the Rho guanine nucleotide exchange factor SGEF and Scribble as important in holding together epithelial cells. This complex forms, in part, through an interaction between Scribble’s PDZ1 domain and SGEF through an internal PDZ motif. PDZ1 offers a place for SGEF to bind (right, in red) and to get an extra grip (right, in yellow) to hold cells together tightly. (Interesting, the three other PDZ domains of Scribble without this specific site cannot bind SGEF securely.) Courtesy: E.Fuentes.

But a crystal structure revealed a new mechanism of PDZ specificity. The SGEF internal motif binds tightly because additional amino acids can latch on to an extra compatible section of Scribble’s PDF domain. The paper was published June 2019 in the Journal of Cell Biology with co-author Rafael Garcia-Mata at the University of Toledo.

The Fuentes lab will work toward a structure of the larger molecular complex with electron microscopy. But the experimental model also opens up a new way to study PDF specificity in more detail using synthetic biology methodologies in collaboration with a research team at Johns Hopkins University.

“We’re now looking at artificial designs of PDZ with the idea of obtaining a deeper understanding of the rules for how nature encodes specificity in a small domain,” Fuentes says. Preliminary work has produced a template protein domain with structural and biophysical properties of a PDZ. “We have a blank slate to design and test the features of specificity,” he says.

In other non-PDZ work from the Fuentes lab, they and their collaborators discovered details of the SrrB two-component signaling system that helps Staphylococcus aureus sense a change from an aerobic to an anaerobic environment.

S. aureus is a common bacterium found in people’s noses and on skin, but inside the body it can turn into a potentially deadly infection. Fuentes struck up a collaboration with a university colleague, Pat Schlievert, who discovered a two-component signaling system important for environmental sensing that allows S. aureus to make the dangerous adaptation to a low oxygen environment.

As they reported in a 2019 paper in PNAS, the Fuentes team found redox active cysteines in a sensor histidine kinase had a big influence on S. aureus pathogenicity.

If there is a theme running through the Fuentes lab, it’s to build an integrative structural biology approach using the tools that best suit the needs of the problem. And to keep an eye on the bigger picture – the biology.

“Structural biology is a part of many studies,” Fuentes says. “It’s not always the central feature. It’s an added method that allows deeper insight into the whole system.”

-Carol Cruzan Morton

Predicting Proteins

Jens Meiler

Published 25 November 2024

Death Metal

Steven Damo

Published 28 April 2024
Context Matters

Bing Chen

Published 30 January 2024

The Crystal Whisperer

Sarah Bowman

Published 29 November 2023
Data in Motion

Nozomi Ando

Published 29 September 2023

The Monstrous Maw

André Hoelz

Published 28 June 2023
Second Takes

Andrea Thorn

Published 28 February 2023

Radical reactions

Yvain Nicolet

Published 31 January 2023
Floppy Physics

Eva Nogales

Published 30 November 2022

Structure of Equity

Jamaine Davis

Published 28 September 2022
Life and Death of a Cell

Evris Gavathiotis

Published 28 July 2022

Follow the glow

Kurt Krause

Published 29 April 2022
Resolution solutions

Willy Wriggers

Published 25 February 2022

Of enzymes and membranes

Ming Zhou

Published 28 October 2021
Step-by-step

Gabrielle Rudenko

Published 26 September 2021

Moving muscle

Montserrat Samso

Published 26 July 2021
Particle catcher

Stefan Raunser

Published 28 June 2021

Designer drugs

Ho Leung Ng

Published 25 February 2021
Right place, right time

Ernesto Fuentes

Published 29 January 2021

Shape-shifting secrets of membranes

James Hurley

Published 27 November 2020
Enzymatic action

Cynthia Wolberger

Published 28 September 2020

Rules of motion

Priyamvada Acharya

Published 31 July 2020
Cosmic Squared

Michael Cianfrocco

Published 27 June 2020

Kaps are Cool

Yuh Min Chook

Published 28 April 2020
Spiraling into focus

Carsten Sachse

Published 29 March 2020

Seeing cilia

Alan Brown

Published 27 February 2020
For the Love of EM

Guy Schoehn

Published 27 January 2020

Protein Puddles

Michael Rosen

Published 16 December 2019
Changing channels

Daniel Minor Jr.

Published 27 September 2019

Listening Tips

Marcos Sotomayor

Published 30 July 2019
Beyond Cool

Published 31 May 2019

Hao Wu

A Higher Order

Published 30 May 2019
Aye Aye Captain

Alexandre Bonvin

Published 29 April 2019

The PARP Family Family

John Pascal

Published 28 February 2019
Frame by frame

Nikolaus Grigorieff

Published 28 January 2019

Predicting Success

Bil Clemons

Published 18 December 2018
Curiouser and Curiouser

Ramaswamy Subramanian

Published 27 November 2018

Rely on This

Sjors Scheres

Published 26 October 2018
Proteins out of bounds

Gerhard Wagner

Published 27 September 2018

Hiding in plain sight

Gaya Amarasinghe

Published 27 July 2018
Jumping Genes

Orsolya Barabas

Published 27 June 2018

Data Whisperer

Karolin Luger

Published 30 May 2018
Flipping the Switch

Jacqueline Cherfils

Published 27 April 2018

Tooling Around

Andrew Kruse

Published 29 March 2018
Comings and Goings

Tom Rapoport, Ph.D.

Published 23 February 2018

Transcriptional Rhythm

Seth Darst

Published 27 January 2018
The Language of Gene Regulation

Daniel Panne

Published 21 November 2017

Not Your Average Protein

James Fraser

Published 23 October 2017
Message Received

Sebastien Granier

Published 24 August 2017

Resistance is Futile

Celia Schiffer

Published 28 July 2017
Twist of Fate

Leemor Joshua-Tor

Published 28 June 2017

Drug Designer

John Buolamwini

Published 30 May 2017
Mathematically Minded

James Holton

Published 28 April 2017

Garbage Out

Kay Diederichs

Published 30 March 2017
Fixer Upper

Brandt Eichman

Published 27 February 2017

Mobilizers

Phoebe Rice

Published 31 January 2017
Escape Artist

Katya Heldwein

Published 19 December 2016

Nature’s Confectioner

Jochen Zimmer

Published 29 November 2016
State of Fusion

Jason McLellan

Published 27 October 2016

Here Be Dragons

Brian Fox

Published 28 September 2016
SBGrid Assumes Ownership of PyMOLWiki

Published 15 September 2016

Pharm Team

Oleg Tsodikov

Published 24 August 2016
Spiro-Gyra

Alejandro Buschiazzo

Published 27 July 2016

Turning the DIALS

Nicholas Sauter

Published 29 June 2016
Pipeline Dreams

Bridget Carragher and Clint Potter

Published 26 April 2016

U-Store-It

The SBGrid Data Bank provides an affordable and sustainable way to preserve and share structural biology data

Published 28 March 2016
Big Questions, Big Answers

Jennifer Doudna

Published 22 February 2016

Not a Structural Biologist

Enrico Di Cera

Published 17 December 2015
Divide and Conquer

Kevin Corbett

Published 19 November 2015

Computing Cellular Clockworks

Klaus Schulten

Published 23 October 2015
Trans-Plant

Gang Dong

Published 26 September 2015

Keep on Moving

James Berger

Published 23 August 2015
Totally Tubular

Antonina Roll-Mecak

Published 27 July 2015

From Disorder, Function

Julie Forman-Kay

Published 29 June 2015
Into Alignment

Geoff Barton

Published 27 May 2015

Two Labs, Many Methods

Michael Sattler

Published 28 April 2015
Picture This

Georgios Skiniotis

Published 20 March 2015

Intron Intrigue

Navtej Toor

Published 20 February 2015
Cut and Paste

Martin Jinek

Published 28 January 2015

Basics and Beyond

Qing Fan

Published 18 December 2014
Bloodletting and Other Studies

Pedro José Barbosa Pereira

Published 25 November 2014

Wire Models, Wired

A brief history of UCSF Chimera

Published 29 October 2014
In Search of…New Drugs

Doug Daniels

Published 30 September 2014

An Affinity for Affinity…and Corals

John C. Williams

Published 29 August 2014
Pete Meyer, Ph.D.

Research Computing Specialist

Published 22 August 2014

Justin O'Connor

Sr. System Administrator

Published 20 August 2014
Carol Herre

Software Release Engineer

Published 15 August 2014

Elizabeth Dougherty

Science Writer

Published 13 August 2014
Andrew Morin, Ph.D.

Policy Research Fellow

Published 11 August 2014

Jason Key, Ph.D.

Associate Director of Technology and Innovation

Published 8 August 2014
Piotr Sliz, Ph.D.

Principal Investigator, SBGrid

Published 1 August 2014

New Kid on the Block

James Chen

Published 29 July 2014
Membrane Master

Tamir Gonen

Published 30 June 2014

The Natural Bridge

Piotr Sliz

Published 13 June 2014
Surprise, Surprise

Catherine Drennan

Published 26 April 2014

Gone Viral

Olve Peersen

Published 20 March 2014
All Who Wander Are Not Lost

Frank Delaglio

Published 24 February 2014

The Raw and the Cooked

Graeme Winter

Published 24 January 2014
Vacc-elerator

Peter Kwong

Published 17 December 2013

Structural Storyteller

Karin Reinisch

Published 15 November 2013
The Fixer

Jane Richardson

Published 28 October 2013

Inside the Box

Mishtu Dey

Published 17 September 2013
Sensing a Change

Brian Crane

Published 16 August 2013

Towards Personalized Oncology

Mark Lemmon

Published 16 July 2013
Brush with Fame

Yizhi Jane Tao

Published 14 June 2013

Toxic Avenger

Borden Lacy

Published 21 May 2013
Pushing the Boundaries

Stephen Harrison

Published 22 April 2013

Strength in Numbers

Joseph Ho

Published 18 March 2013
One Lab, Many Methods

Wesley Sundquist

Published 12 February 2013

Unplanned Pioneer

Tim Stevens

Published 15 January 2013
From Actin to Action

Emil Pai

Published 11 January 2013

Unstructured

A Brief History of CCP4

Published 12 December 2012
Stop, Collaborate and Listen

Eleanor Dodson

Published 5 November 2012

X-PLORer

Axel Brunger

Published 1 October 2012
Share the Wealth

Zbyszek Otwinowski

Published 22 August 2012

Unraveling RNA

Anna Pyle

Published 18 July 2012
Sharper Image

Pawel Penczek and SPARX

Published 4 June 2012

Creative Copy Cat

Pamela Bjorkman

Published 25 April 2012
Charm and Diplomacy

Gerard Kleywegt

Published 7 March 2012

From Curiosity to Cure

Marc Kvansakul

Published 13 December 2011
The Lure of the Sandbox

Paul Emsley and Coot

Published 15 October 2011

Springsteen, Tolkien, Protein

Alwyn Jones and Frodo

Published 17 June 2011
Structures Solved Simply

Paul Adams and Tom Terwilliger on Phenix

Published 2 June 2011

Playing the Odds

Randy Read and Phaser

Published 19 May 2011
Escape from the Darkroom

Wolfgang Kabsch and XDS

Published 19 May 2011

Crystallography for Kids

Lynne Howell

Published 17 May 2011
Better, Faster, Stronger, More

Victor Lamzin and ARP/wARP

Published 17 May 2011

Side-Track to Success

Ning Zheng

Published 13 May 2011

Predicting Proteins

Death Metal

Context Matters

The Crystal Whisperer

Data in Motion

The Monstrous Maw

Second Takes

Radical reactions

Floppy Physics

Structure of Equity

Life and Death of a Cell

Follow the glow

Resolution solutions

Of enzymes and membranes

Step-by-step

Moving muscle

Particle catcher

Designer drugs

Right place, right time

Shape-shifting secrets of membranes

Enzymatic action

Rules of motion

Cosmic Squared

Kaps are Cool

Spiraling into focus

Seeing cilia

For the Love of EM

Protein Puddles

Changing channels

Listening Tips

Beyond Cool

Hao Wu

Aye Aye Captain

The PARP Family Family

Frame by frame

Predicting Success

Curiouser and Curiouser

Rely on This

Proteins out of bounds

Hiding in plain sight

Jumping Genes

Data Whisperer

Flipping the Switch

Tooling Around

Comings and Goings

Transcriptional Rhythm

The Language of Gene Regulation

Not Your Average Protein

Message Received

Resistance is Futile

Twist of Fate

Drug Designer

Mathematically Minded

Garbage Out

Fixer Upper

Mobilizers

Escape Artist

Nature’s Confectioner

State of Fusion

Here Be Dragons

SBGrid Assumes Ownership of PyMOLWiki

Pharm Team

Spiro-Gyra

Turning the DIALS

Pipeline Dreams

U-Store-It

Big Questions, Big Answers

Not a Structural Biologist

Divide and Conquer

Computing Cellular Clockworks

Trans-Plant

Keep on Moving

Totally Tubular

From Disorder, Function

Into Alignment

Two Labs, Many Methods

Picture This

Intron Intrigue

Cut and Paste

Basics and Beyond